How Molecular Structure Influences Potency of a Therapeutic Biologic

Merck KGaA
Darmstadt, Germany
February 7, 2019
Daniel Galbraith, Ph.D.
Piotr Kaczmarek, Ph.D.
Agata Burzawa, M.S.
How molecular
structure influences
potency of a
therapeutic biologic
1

The life science business of
Merck KGaA, Darmstadt, Germany
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2 How molecular structure influences potency of a therapeutic biologic | 07.02.2019

Merck KGaA
Darmstadt, Germany
Monoclonal antibodies are a rapidly
expanding sector.
These drugs are complex in their structure
and their mode of actions.
Today we will have a closer look at one of
these drugs: Humira® (Adalimumab).
The focus of this presentation is glycans. We
will look at how to choose an analytical
method and how changes to these glycan
molecules affect the biological activity of
the drugs.
Introduction

Merck KGaA
Darmstadt, Germany
N-Glycan Profiling
and Impact of the
de-N-glycosylation on the
structure of the IgG1
monoclonal antibody
Piotr Kaczmarek, Ph.D.
Principal Scientist, Testing R&D Services
4

Monoclonal antibodies (mAbs) are:
- A very dynamically growing class of therapeutic
proteins
- 570+ drug candidates in clinical trials
(https://www.antibodysociety.org/tag/approved-
antibodies/)
- IgG1 is one of the most commonly used type of mAb
Monoclonal Antibodies
VL
CL
VH
CH1
CH2
CH3

Typical IgG1 mAb consist of:
- 2 identical Light Chains (LCs)
o 1 Variable Domain (VL)
o 1 Constant Domain (CL)
- 2 identical Heavy Chains (HCs)
o 1 Variable Domain (VH)
o 3 Constant Domains (CH1, CH2, and CH3)
- Multiple intra- and interchain disulfide bonds
- 1 N-glycosylation site in the Fc domain of HC
Structure of Typical IgG1 mAbs
VL
CL
VH
CH1
CH2
CH3
Fab
Fc

Typical IgG1 mAb consist of:
- 2 identical Light Chains (LCs)
o 1 Variable Domain (VL)
o 1 Constant Domain (CL)
- 2 identical Heavy Chains (HCs)
o 1 Variable Domain (VH)
o 3 Constant Domains (CH1, CH2, and CH3)
- Multiple intra- and interchain disulfide bonds
- 1 N-glycosylation site in the Fc domain of
HC
Structure of Typical IgG1 mAbs
VL
CL
VH
CH1
CH2
CH3
Fab
Fc

Presence and structure of the Fc N-glycans may impact:
- Structure of the monoclonal antibody
- Activity and/or potency of the monoclonal antibody
Function of Fc N-Glycans

Performing N-glycan profiling is important

Typically, two types of information are expected:
- Relative abundance of individual N-glycans
- Identity of N-glycans
Strategies for N-Glycans Profiling

Strategy:
- Label-free glycans
- Labelled glycans

Strategy:
- Labelled glycans

Strategy:
- Labelled glycans
o UHPLC/UPLC-FLD
o CE-LIF

N-Glycan Profile
of Adalimumab

Case Study: Adalimumab
Humira (Adalimumab) is:
- A recombinant human IgG1 monoclonal antibody specific for human tumor
necrosis factor (TNF)
- Used for treatment of:
o Rheumatoid Arthritis
o Juvenile Idiopathic Arthritis
o Psoriatic Arthritis
o Adult Crohn’s Disease
o Pediatric Crohn’s Disease
www.Rxlist.com
o Ulcerative Colitis
o Plaque Psoriasis
o Hidradenitis Suppurativa
o Uveitis
Clinical applications

Case Study: Adalimumab
PNGase F Labelling
Glycan structure

Case Study: Adalimumab (UHPLC/UPLC-FLD)
Relative abundance of each N-glycan

Case Study: Adalimumab (UHPLC/UPLC-FLD with On-Line MS
Detection)

Detection)

Detection)
Relative abundance and identification of each glycan

Case Study: Adalimumab (CE-LIF)
Relative abundance and identification of each glycan

Case Study: Adalimumab (UHPLC/UPLC vs. CE)

Impact of Fc N-
glycosylation on
the structure of
IgG1 monoclonal
antibody

To determine the impact of the presence of N-glycans,
compare structures of:
- Native IgG1 monoclonal antibody
- IgG1 monoclonal antibody de-N-glycosylated under non-denaturing
conditions
Case Study: Design

To determine the impact of the presence of N-glycans,
compare structures of:
- Native IgG1 monoclonal antibody
- IgG1 monoclonal antibody de-N-glycosylated under non-denaturing
conditions
We want to:
- Understand the nature of the change
- Localize the site where the change occured
Case Study: Design

Analytical methods for analysis of protein structure:
- NMR
o Typically requires higher sample concentration
o Not a routine measurements for mAb
- X-Ray crystallography
o Depends on obtaining high quality protein crystals
Case Study: Design

Analytical methods for analysis of protein structure:
- NMR
o Typically requires higher sample concentration
o Not a routine measurements for mAb
- X-Ray crystallography
o Depends on obtaining high quality protein crystals
- Hydrogen-Deuterium Exchange-Mass Spectrometry (HDX-MS)
o Requires low amount of the sample (50 µL of 3.0 mg/mL mAb solution
is sufficient)
o Suitable for routine measurements of IgG mAbs
Case Study: Design

Hydrogen-Deuterium Exchange-Mass Sectrometry (HDX-MS) is:
- One of the most advanced techniques for measuring the Higher Order Structure of proteins
- Based on spontaneous exchange of hydrogen atoms in labile positions in proteins with
deuterium atoms from the surrounding solvent
o With peptide backbone amide hydrogens being of the main interest due to variations in
their chemical and physical environment induced by protein structure (solvent
accessibility, hydrogen bonding, etc.)
- A comparative technique, which can be used for:
o Comparing multiple lots of the same material
o Comparing stressed vs. non-stressed samples
o Performing antigen mapping
- Recommended literature: D. Houde and J.R. Engen, Methods Mol. Biol. 988 (2013) 269-289
Case Study: Design

Case Study: Design of the HDX-MS Workflow
D. Houde and J.R. Engen, Methods Mol. Biol. 988 (2013) 269-289

NISTmAb (NIST RM 8671) is:
- A humanized IgG1 monoclonal antibody
- Fully characterized universal Reference Material
- Designed for:
o Evaluation and optimization on analytical methods
SILu™Lite SigmaMAb Universal Antibody Standard (Sigma, Cat# MSQC4) is:
- A human IgG1 monoclonal antibody
- Fully characterized universal Reference Material
- Designed for:
o Evaluation and optimization on analytical methods
Case Study: NISTmAb and SILuLite SigmaMAb

Case Study: Experiment
VL
CL
VH
CH1
CH2
CH3
VL
CL
VH
CH1
CH2
CH3
vs.

VL
CL
VH
CH1
CH2
CH3

VH
CH1
CH2
CH3

VH
CH1
CH2
CH3
0%
5%
10%
15%
20%
25%
30%
35%
0 5 10 15 20 25 30
RelativeDeuteriumUptake(%)
Time (min)
HC(5-19)
native
deglyco
0%
2%
4%
6%
8%
10%
12%
14%
16%
0 5 10 15 20 25 30
Time (min)
HC(83-94)
native
deglyco

VH
CH1
CH2
CH3
0%
1%
2%
3%
4%
5%
6%
7%
8%
9%
10%
0 5 10 15 20 25 30
Time (min)
HC(149-158)
native
deglyco
0%
1%
1%
2%
2%
3%
3%
4%
4%
0 5 10 15 20 25 30
Time (min)
HC(183-188)
native
deglyco

VH
CH1
CH2
CH3
0%
5%
10%
15%
20%
25%
30%
35%
40%
0 5 10 15 20 25 30
Time (min)
HC(238-243)
native
deglyco
0%
5%
10%
15%
20%
25%
30%
0 5 10 15 20 25 30
Time (min)
HC(244-255)
native
deglyco

VH
CH1
CH2
CH3
0%
5%
10%
15%
20%
25%
30%
0 5 10 15 20 25 30
Time (min)
HC(244-255)
native
deglyco
0%
5%
10%
15%
20%
25%
0 5 10 15 20 25 30
Time (min)
HC(256-264)
native
deglyco

VH
CH1
CH2
CH3
0%
5%
10%
15%
20%
25%
0 5 10 15 20 25 30
Time (min)
HC(372-383)
native
deglyco
0%
5%
10%
15%
20%
25%
30%
35%
0 5 10 15 20 25 30
Time (min)
HC(436-449)
native
deglyco

Conclusion:
- De-N-glycosylation leads to increased deuterium uptake in CH2 domain
(residues 238-255)
- The increased deuterium uptake suggests unfolding or change of
conformation within CH2 domain
Case Study: Conclusions

Conclusion:
- De-N-glycosylation leads to increased deuterium uptake in CH2 domain
(residues 238-255)
- The increased deuterium uptake suggests unfolding or change of
conformation within CH2 domain
Relevance:
-Based on X-ray crystallography, this region was identified as an anchoring point of
FcγRIII receptor binding (Radaev et al., J. Biol. Chem. 276 (2001) 16469-16477).
-Upon de-N-glycosylation, similar disturbance in the CH2 domain of mAb was
observed for:
o SigmaMAb (unpublished data)
o Undisclosed IgG1 monoclonal antibody reported in D. Houde et al., Anal. Chem. 81
(2009) 2644-2651
Case Study: Conclusions

Merck KGaA
Darmstadt, Germany
Piotr described multiple methods to
investigate glycan structures.
Some methods such as Capillary
Electrophoresis are suitable for high
throughput samples.
Other methods such as Mass Spectroscopy
are more suited to detailed examination
Knowing the glycan structure is the start
now we need to relate this to the biological
functions.
Glycan Analytics

Merck KGaA
Darmstadt, Germany
Agata Burzawa, M.S.
Senior Scientist, Global Operational Development Services
Influence of galactosylation
on Fc-mediated binding and
functional properties of
adalimumab

Functional Impact of glycosylation profile
Structure
Biological
activity
Binding
Core fucose : HIGH
❑ Impacts FcγIIIa binding and ADCC
Galactose: MODERATE
❑ Differences may impact C1q binding and CDC
High mannose
❑ Increased ADCC and reduced CDC
❑ More rapid clearance
Sialyation NN-acetylneuraminic acid: MODERATE
❑ May impact PK
❑ May impact Fc functionality

Study Overview
Concentration
measurement
Digestion
Purification
Structure and
function
analysis
β-galactose removal from
N-glycans using β-1,4
galactosidase
Purification of antibody
samples after enzymatic
digestion using Nab™
Protein A spin column
N-Glycan profile modification

Methods – Structure / function investigation
Structure
N-glycan analysis (CE-
LIF)
Function
Binding
C1q
binding
(ELISA)
FcyIIIa
binding
(SPR)
Biological
activity
CDC ADCC
To evaluate the criticality of glycan structure on antibody Fc-region mediated effector functions, we
analyzed the N-glycan profile of the following:
- Stock Adalimumab (untreated)
- Adalimumab mock control
- Adalimumab enzyme treated
This was achieved using a highly resolving N-glycan assay, in combination with binding and cell-
based assays.

N-glycan analysis by capillary electrophoresis coupled with
laser induced fluorescence detector (CE-LIF)
60 minute protocol
• High speed sample separation
• 96-well plate format
• High level of reproducibility
• High resolution of N-glycan separation obtained within 5 minutes
• Automated glycan identification
Prepare
reagents
and stock
solutions
Denature
proteins
Release
the N-
Glycans
Label the
glycans
Separate
the
glycans
Analyse
and
identify
the
glycans

N-glycan analysis of mock control and enzyme treated
adalimumab using CE-LIF.
Adalimumab mock
control
Adalimumab enzyme
treated
N-
Glycan
Type
Area (% of total glycan species
detected)
Adalimumab
mock control
Adalimumab
enzyme treated
G0F 73.562 86.861
G1F 11.806 0.611
G1’F 3.74 Not detected
G2F 0.913 Not detected
Successful digestion of terminal β-galactose

Antibody dependent cellular cytotoxicity (ADCC)
NK cell degranulation
& lysis of target cell
Effector Cell (e.g. NK)
attachment via FcɣR)
mAb binding target
antigen on cells
Cell line expressing target antigen
• ADCC was performed using a recombinant mTNFα CHO-K1 target cell line (Promega).
• Fresh human peripheral blood mononuclear cells (PBMCs) were isolated from healthy blood donors by
gradient centrifugation.
• To detect ADCC activity of Adalimumab, the target cell line was treated with a sample dilution series
followed by the addition of PBMCs.
• After overnight incubation, the ADCC activity was detected using a luminescence based end-point reagent.
• Data generated was reported as relative potency (RP) values.
PROCEDURE

FcγIIIa Binding by SPR
Fc Region
FcɣRIIIA (V)
RU
Time (S)
The binding affinity of all samples for recombinant human FcɣIIIa protein was determined using surface
plasmon resonance (SPR) technique on a Biacore™ T200 instrument (GE Healthcare).
Multi cycle kinetics for each sample was performed using multiple different concentrations of Adalimumab
as the analyte flowing over a directly immobilised FcγIIIa surface (CM5 chip) and the resulting KD value
was established using Biacore software.

Relationship between FcγIIIa Binding and ADCC activity
Parameter
Adalimumab mock
control
Adalimumab
enzyme treated
ka (1/Ms) 2.06 x 10 5 6.79 x 104
kd (1/s) 0.03936 0.01639
KD (M) 1.913 x 10-7 2.416 x 10-7
Relative KD (%) 100 79.2
Parameter
Adalimumab mock
control
Adalimumab
enzyme treated
Potency NA 43.9%
EC50 0.006 0.011
A 56.1% reduction in ADCC activity was observed for enzyme treated
sample. A corresponding decrease was also detected in FcγIIIa receptor
binding represented by a 20.8% reduction in affinity (KD).
FcγIIIa binding ADCC
Adalimumab concentration [ug/mL]

Complement dependent cellular cytotoxicity (CDC)
• The CDC assay was performed using a recombinant mTNFα CHO-K1 target cell line
(Promega).
• Cells were treated with sample dilution series in the presence of pooled human serum
complement for 2h at 37ºC.
• The CDC activity was measured using a luminescence based end point reagent.
• Data generated was reported as relative potency (RP) values.
PROCEDURE
mAb binding to
target antigen
Lysed target cell
Cell line expressing target antigen
mAb-C1q Binding
& MAC formation

Binding to C1q protein
• Samples from Adalimumab dilution series
were added to the ELISA plate.
• After overnight incubation at 4ºC, 2µg/mL of
human C1q was added to the wells
• bound C1q protein was detected using an
anti-C1q-HRP conjugated antibody followed
by addition of TMB substrate.
• The reaction was stopped with 1M HCl, and
the absorbance was read at 450nm.
• Data generated was reported as relative
binding potency (RP) values.
PROCEDURE
Adalimumab
C1q protein
Detection
antibody
TMB

Relationship between C1q Binding and CDC activity
Parameter
Adalimumab mock
control
Adalimumab
enzyme treated
Potency NA NA
EC50 0.835 3.462
Parameter
Adalimumab mock
control
Adalimumab
enzyme treated
Potency NA 59.3.1%
EC50 0.457 0.598
A significant decrease in CDC activity for enzyme treated sample
was observed. Results showed that reduced Fc-galactosylation
decreases CDC activity through lower binding affinity to C1q.
C1q binding CDC

Results summary
In summary :
• Adalimumab mock control contained 16.4% of galactosylated species (G1F, G2, G2F). After enzyme
treatment , the content of galactosylated species decreased to 0.6%, which was associated with;
• An overall reduction in binding affinity to FcγIIIa (20.8% reduction in KD value) and C1q protein
(75.9% reduction in Ec50 value).
• A 56.1% reduction in ADCC activity (RP)
• A 40.7% reduction in CDC activity (RP)
100 100 100 100
79,2
43,9
24,1
59,3
0
10
20
30
40
50
60
70
80
90
100
FcγIIIa
binding
ADCC C1q ELISA CDC
Relativeactivity[%]
Adalimumab mock
control
Adalimumab enzyme
treated

Summary
Clone selection
Media and feed
supplements (USP
development)
Process
development and
purification (DSP)
Production and
scale up
QC testing
and comparability
studies
• Our data confirmed that glycan structure is a critical quality attribute of Adalimumab, and highlights
the importance of investigating glycan composition during the development of new therapeutic
antibodies.
• The results obtained were consistent with literature reports showing that a lack of terminal
galactose disturbs the effector functions of examined antibodies.
• Fast N-Glycan analysis is an alternative, rapid, method of glycosylation pattern assessment which
may be very useful in providing information during critical stages of new molecule development.

Merck KGaA
Darmstadt, Germany
Glycan Analytics coupled with Biological
function can be designed for each drug
Both ADCC and CDC activity have
measurable changes when even small
glycan changes are made
High throughput Glycan analytics is possible
for testing samples
Other applications can be examined –
Talk to the team
Conclusions

How Molecular Structure Influences Potency of a Therapeutic Biologic

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