Understanding your monoclonal antibody sounds simple; however, to get a comprehensive understanding of the quality of your molecule, one must take a holistic view. How do structural variants, post translational modifications, and the manufacturing process affect your molecule? The application of orthogonal methods for early phase mAb and biosimilar production, deliver detailed understanding of the mAb structure-function relationship, increasing the success of regulatory approval and speed to clinic. This webinar will use case studies to illustrate why characterizing the physio-chemical and structural attributes in conjunction with the biological activity, will mitigate risks associated with the development of complex biologics. In this webinar, you will learn: • Range of analytical and bioanalytical capabilities required, and the value of a holistic approach by: o Gaining a comprehensive understanding of your mAb molecule o Enhancing appreciation of the manufacturing process o Understanding parameters that impact quality attributes and stability • Utilization of advanced technology to improve: o Understanding of relationship between structure and function

1. Merck KGaA
Darmstadt, Germany
Pamela Hamill, Ph.D.
Principal Scientist, Global Operational Development Services

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

3. 1. Characterisation of therapeutic antibodies
 What, why, when
 Benefits of early use of PC approaches to understand your product
2. Complexities of monoclonal antibodies (mAbs) and their characterisation
3. Approaches to characterisation of mAb physico-chemical and structural attributes
4. Functional characterisation of mAbs
Webinar Outline
3

4. Product Characterization
4
Physico-
chemical
analysis
Biological
activity
Product
specific
impurities
Product
Characterisation
▪ Product characterisation lays
the foundations for successful
biologic drug development
▪ In depth product
understanding facilitates
process design to ensure the
required product safety,
purity, identity and potency
profiles are achieved

5. 5
Analytics and characterisation answers crucial questions
Structural information
• Peptide sequence
• Molecular mass
• Glycan Structure
• Charge variant profile
• Aggregates/Impurities
• Chemical modifications
Biological activity
• Does it bind as expected to the
target and what is the strength of
the interaction?
• What biological activity does it have?
• Does it engage immune system
components (e.g. NK cells,
complement, macrophages) to bring
about effector functions?
Structure
Binding
Biological
Activity
(Potency)
How does my
drug function?
What are the
physical and
structural
attributes of
my drug?

6. Product Characterisation plays a critical role in early development stages
Drug developers are increasingly investing in analytics early to maximize risk mitigation
c
•Clone selection
•USP and DSP
optimisation
•Establish CQA
Establish
product
Reference
standard
Subset of PC
methods used
for Lot release
& stability
Support comparability
studies following
changes in
manufacturing
6

7. ▪ Characterisation of physicochemical properties and biological activity satisfies regulatory expectations
(ICH Q6B), but use of powerful and high resolution methods is often not applied routinely at early stages
of development
▪ If
Value Proposition:
Product Characterization in early stage development
7
Benefits of early
investment in
product
characterisation
activities
▪ Better understanding of the product, its structure-function relationship and
heterogeneity is achieved earlier, helping to identify critical quality attributes
▪ Insight into how process conditions impact product quality as well as the sources
and impact of heterogeneity and how these can be mitigated, is also gained
sooner... Informs process development
▪ Early use of selected PC methods adds additional insight related to attributes
predicted to be relevant to the intended mode of action
Reduce risk of
biologics
Development:
fewer
‘unknowns’

8. Analytical characterisation contributes to incorporating QbD
principles into the development process
8
Process
development
Assess quality
attributes
Refine
Process
Assess quality
attributes
ANALYTICAL
CHARACTERISATION
ANALYTICAL
CHARACTERISATION
START

9. ▪ The size and complexity of mAbs, and their potential for
considerable heterogeneity, means they are a complex
scenario for product characterization
▪ Since each mAb domain can interact with different
binding partners and can mediate a variety of biological
functions, extensive physico-chemical and structural
analysis must be complemented with in-depth
characterization of the biological binding and functional
activities
▪ Heterogeneity will occur, but using the right methods this
can be characterised and analysed to check the product
variability does not hinder the required functions
Challenges of characterizing complex molecules:
Monoclonal antibody products (mAbs)
9
Fragment
antigen binding
(Fab)
Fragment
crystallisable
(Fc)

11. characterization
of mab products:
physico-chemical and
structural attributes

12. Sources of mAb heterogeneity and possible consequences
12
Attribute
Source of heterogeneity/
Impurity
Possible clinical Implications
Primary Structure
Post-translational
modifications (PTMs)
Primary aa sequence
Glycan Structure, e.g.
• Sialic Acid
• Fucosylation
Deamidation/Oxidation
Altered binding to target antigen, effector
components, changes to HOS, Loss of
stability
Impact on binding to FcɣR/FcRn/C1q and
subsequent effector functions
Immunogenicity and effector functions
G0 isoform fucosylation impacts ADCC
activity
Potential impact of target binding if
Met/Asn residues in antigen binding region
Higher Order
Structure (HOS)
Di-sulphide bridge structure
(IgG2/IgG4)
Loss of HOS required for optimal
functionality
Impurities Aggregation
Potential to increase immunogenicity
Potency may be affected
Variants Charge Variants
Altered binding affinity to FcɣR/FcRn
Potency may be affected

13. 131313
 Mitigation of risk through directed PC
Monitoring the structural properties and the natural heterogeneity of your product using the right
methods can demonstrate what attributes can be variable to retain the required functional activities as
well as provide assurance that the process consistently generates product with similar profiles of
hetergeneity
Physicochemical/Structural variability and consequences
Sources of
structural
variability
RISKS
Differences or changes in physico-chemical
properties, post-translational modification and
higher order structure can significantly affect
product quality attributes and hence present
risk to:
Efficacy Safety StabilityPurity
Examples;
N-Glycosylation
C-terminal lysine
Oxidation
Deamidation-
-

14. Typical mAb physicochemical and structural characterisation:
Comprehensive analysis & understanding of links betweeen structural properties and required quality
attributes reduces risk during product lifecycle
S
Primary aa
sequence
Glycan profiling – N-glycan map (critical for
understanding FcɣRIIIA binding and ADCC
activity)
Disulphide bond analysis (especially for
IgG2/IgG4 mAbs):variability can impact
stability and biological activityPurity (reduced and non-
reduced)
Charge profile (cIEF): identity and
stability testing...charge variants
Intact MW (LC-
MS)
Determination of
monomer/aggregate and
fragment content
De-N-glycosylated
MW
Peptide mapping (LC-MS/MS
to identify and quantify
PTMs)
14

15. Molecular weight analysis by LC-MS
15
Intact MW De-N-glycosylated MW
Antibody Theoretical MW
Averaged
determined MW
Theoretical MW
Averaged
determined MW
SigmaMab 146,818.5* 146,823 ± 1 143,767.7 143,767.7 ± 0.2
Cetuximab 152,649.6† 152,649 ± 2 149,598.8 149,599.9 ± 0.6
Adalimumab Unknown** 148,823 ± 1 Unknown** 145,189.0 ± 0.3
* Calculated for the G0F/G1F glycoform, †Calculated for G0F/G1F and G2FGalSA/ G2FGalSA glycoform,* * No access to primary sequence
Data obtained by Waters Acquity H Class UPLC/Waters Synapt 2G Si MS confirms
molecular theoretical MW from drug database information
MS analysis to confirm the fundamental property of the expected MW based on amino acid
sequence and peptide backbone in the absence of glycosylation

16. Product Impurity
Capillary Electrophoresis (CE-SDS)
16
Purity and extent of variability of size variants can be confirmed using Capillary SDS electrophoresis in
both the reduced and non-reduced states
Offers high resolution solution for separation of heavy and light chains contaminants as well
as glycosylated and non-glycosylated forms
Adalimumab-non-reduced Adalimumab-reduced

17.  Determining the relative abundance of Higher and Lower Molecular Weight Components (HMWCs
and LMWCs), representing aggregates and fragments of the mAb, as compared with the
monomeric form, is an important quality determinant
 A sensitive method is necessary to identify product aggregates, since this is a common tendency
for highly purified and concentrated mAb therapies
Product Impurity
Aggregation
17
Increased Immunogenic potential
Potential impact on other CQA, e.g.
potency (reduced availability to target)
Changes to the desired monomeric state of therapeutic mAbs can have significant
effects on product quality.

18. Product Impurity
Profiling to reveal product size variability
18
Product Aggregation
▪ Size exclusion chromatography with UV detection to detect changes in therapeutic mAb aggregation
profiles following stress of molecules in two different ways was performed
▪ Relative abundances of monomeric peak, HMWC and LMWC for Cetuximab and Adalimumab:
Adalimumab
Chromatograms
(Two flow rates)
Size-exclusion chromatography/UV detection coupled with Dynamic Light scattering enables
greater accuracy in quantification of aggregates
Adalimumab

19. Electropherogram data generated using BioReliance® methodology indicates a more
complex charge profile for Cetuximab as compared with Adalimumab
Charge profile by cIEF: Adalimumab and Cetuximab
19
Adalimumab Cetuximab
Protein Simple iCE3
with autosampler
▪ Basic or acidic charge variants are common alongside the main species in mAb products
▪ Capillary isoelectric focusing (cIEF) offers high throughput/ resolution analysis of charge variants
which must be characterised and controlled since product immunogenicity and potentially other CQA
may be affected by variability

20. Peptide mapping: multi-purpose characterisation tool
20
Total ion current chromatogram
MS/MS data
Peptide mapping of Cetuximab by LC-MS/MS
Peptide mapping is recommended to identify proteins or quantify PTMs
Multiple enzymes can be used to obtain the necessary level of peptide coverage of the protein
Waters Acquity H Class UPLC system/
Waters Synapt 2G Si Mass Spectrometer

21. N-Glycan Profiling
Heterogeneity in carbohydrate PTMs
21
↓↑ Effector functions:
• ADCC
• CDC
• ADCP
NK
MΦ
FcɣR & C1q
Binding
▪ mAb glycan structure can be complex owing to possible additions
of fucose, galactose, mannose, sialic acid and N-
acetylglucosamine moieties in eukaryotic cells
▪ Variability in glycan structure is a major cause of heterogeneity
and one of the better known examples of how structural
differences in PTM can alter biological activity, e.g1.
▪ Afucosylation leading to ↑ FcɣRIIIA binding/ADCC activity
▪ Galactosylation leading to ↑ binding to C1q /CDC activity
▪ Differences in Immunogenicity, and PK/PD properties
▪ Ensuring product quality relies on an advanced glycan analysis
method for assessing whether the glycan profile and
heterogeneity of your antibody product gives rise to the desired
Fc effector functions
1. Reusch D, Tejada ML. Fc glycans of therapeutic antibodies as critical quality attributes. Glycobiology 2015; 25(12):1325–34

22. Glycan Profiling
N-glycan analysis of Adalimumab
22
Representative data generated using Fc N-glycan map by LC-MS assay performed at
BioReliance®
Sensitive assignment of Glycan structure facilitates understanding of the binding
characteristics between Fc regions and immune components and the resulting
effector functions
Waters Acquity H Class UPLC system/
Waters Synapt 2G Si Mass Spectrometer
Mass
Spec.
Fluorescence
Detection

23. ▪ The physicochemical and structural characteristics
are fundamental to the biological activity of the
molecule.
▪ Analysis of structural attributes must be
complemented with sensitive binding and cell
based assays which best reflect the possible
mechanisms of action
Correlating physicochemical and structural properties with functional
analysis
23
Biological
function
characterisation
Structural
Characterisation &
physicochemical
properties
Product
understanding
and reduction
of risk

25. Functional
characterization
of mab products:
binding and biological
activity

26. Components of Functional Characterisation
Binding
Activity
Biological activity
(function)
Functional
characterisation
AIM:
▪ Determine significance of structural attributes and
variability on drug’s biological function
▪ Biosimilars: functional significance of differences
between molecules?
ASSAY CRITERIA:
▪ Sensitive
▪ Cell based... reflect modes of action of drug in vivo
26

27. 27
Cell expressing
Target antigen
Fab region of
antibody binds
to antigenic
target
Therapeutic
effect
Fc region can bind to
FcɣR on immune
effector cells or C1q
complement protein
FcɣR-
expressing
Effector Cells
(NK, MΦ,
neutrophils)
C1q
Antibodies can work in a
variety of ways… clinical
efficacy typically depends
on the combination of
biological functions
mediated by both the Fab
and Fc regions

28. mAb Functional Characterization: Fab and Fc mediated activities
28
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)
Understanding the possible functions and what your antibody is intended to do, enables
selection of the most appropriate characterisation methods for your product

29. CELL BASED FUNCTIONAL ASSAYS
 Critical to use assays that;
 reflect possible modes of actions in vivo
 sensitive detection of biological activity.
 Can be challenging for mAbs!
 multiple assays are needed,
 primary cells are often needed,
 insight and experience are critical to choose
and develop the right assays for your product.
e.g for lot release and stability
 Partnering with outsource provider is often a
good strategy to access the experience and
instrumentation needed to deliver meaningful
cell based assays
BINDING ASSAYS
Surface Plasmon Resonance (SPR) has become the
leading technology to analyse antibody binding
interactions mediated by both the Fc and Fab
regions.
▪ Real-time information on the binding (association
or ‘ON’) and disassociation (OFF) rates between
interacting molecules= KINETIC ANALYSIS (more
informative than ELISA)
▪ Label free, rapid and low volume requirements
Elucidating functional activity of mAbs
29
RU
Time (S)
KD = kd / ka
BINDING AFFINITY
ka kd

30. Case Study - Adalimumab
Characterization of mAb products; using knowledge of intended MOA
30
Most significant MOA
(Fab mediated)
• Adalimumab binds to soluble TNF (sTNF)
• Prevents TNF binding to receptors
• Inhibits pro-inflammatory/pro-
apoptotic effects of TNF-very
important in rheumatoid
arthritis/psoriasis
Other MOA of Adalimumab
Fc region mediated after
binding to cell membrane
bound TNF (tmTNF)
• ADCC
• CDC
Well
characterised and
successful target
for biosimilar
development
IgG1 mAb
therapeutic

31. Case Study - Adalimumab
Fab Binding and biological activity
31
Fab
Region
Mean KD =
231.2 pM
Adalimumab-TNF interaction
• A binding assay enabling kinetic, real-time analysis of the
binding interaction between the Fab region of Adalimumab its
target antigen, TNF-, was developed using SPR
• The assay uses a capture approach to immobilize Adalimumab,
followed by kinetic binding analysis of multiple concentrations
of the analyte (TNF-α) in order to generate a dissociation
constant (KD) value

32. Case Study - Adalimumab
Biological consequence of TNF-binding: TNF Neutralization Activity
32
Add recombinant human TNF solution and incubate with
adalimumab serial dilutions for 30 minutes
Seed L929 target cells and addition of pre-incubated
adalimumab and TNF treatments
Incubation (17±1 hours at 37 °C in humidified CO2 incubator)
Add CytoTox-GLO™ cytotoxicity reagent for quantification of cell
death
Measure luminescence; construct dose response curves then
assess parallelism and RP
Preparation of adalimumab serial Dilutions
BioReliance® relative potency assay can
measure between 50% to 200% relative TNF
neutralizing activity (dose dependent increase
in survival of TNF sensitive cells when treated
with Adalimumab)

33. Case Study - Adalimumab
Characterization of Fc binding interactions by SPR
33
FcɣRI
RU
Time (S)
FcɣRIIIA (V)
RU
Time (S)
FcɣRIIIB
RU
Time (S)
FcɣRIIA
RU
Time (s)
FcɣRIIB
RU
Time (s)
FcɣRIIIA (F)
Fc
Region
FcRN
RU
Time (s)
FcRn
KD: FcɣRI > FcɣRIIIA(V) > FcɣRIII(F) > FcɣRIIIB
Time (S)

34. Linking structure and function;
Antibody dependent cellular cytotoxicity (ADCC)
34
ADCC assays measure the cell killing induced by mAb binding to cell associated target antigen and
subsequent binding of immune effector cells (via Fcɣ receptors) leading to degranulation and
destruction of the target cell.
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
Affinity for FcɣRIIIA
receptors (SPR) ??
Fc Glycan profiling:
fucosylation levels??
A B
C
Impact on in vitro
ADCC activity??

35. Case Study - Adalimumab
Characterization of ADCC activity
35
Preparation of adalimumab dilutions & fresh human PBMC
Treatment with Adalimumab serial dilutions
Incubation at 37° C
Measure luminescence; construct dose response curves then
assess parallelism and RP
Seed the membrane-bound TNF-expressing target cells
Add CytoTox-GLO™ reagent to quantify the cell death
Treatment with primary human PBMC
Sample with increased
RP (1.29)
Sample with decreased
RP (0.66)

36. Linking structure and function;
Complement dependent cytotoxicity (CDC)
36
CDC assays measure the cytotoxic effect induced by mAb binding to cell associated target antigen and subsequent Fc-
mediated binding to C1q complement protein, leading to formation of the membrane attack complex (MAC) and lysis of
the bound cell.
mAb binding to
target antigen
Lysed target cell
Cell line expressing target antigen
mAb-C1q Binding
& MAC formation
Fc Glycan profiling:
Terminal galactose ??
A B Affinity for
C1q ??
C
Impact on in vitro
CDC activity??

37. C1q binding interaction by ELISA
37
Therapeutic
mAb
C1q protein
Detection antibody:
Anti-C1q-HRP
TMB
Adalimumab
Cetuximab
Relative C1q binding potency of Adalimumab
samples (70%, 100, 130%)
Comparison of C1q binding of Adalimumab
and Cetuximab: Adalimumab shows a higher
binding affinity than Cetuximab, consistent with
its ability to induce CDC in an in vitro assay and
observations in the literature
Relative C1q binding potency of Cetuximab
samples (80%, 100, 135%)

38. Case Study - Adalimumab
Characterization of CDC activity
38
Seed the membrane –bound TNF overexpressing cells
Treatment with adalimumab serial dilutions and complement
Incubation (2 hours at 37 °C in humidified CO2 incubator)
Measure luminescence; construct dose response curves then
assess parallelism and RP
Preparation of adalimumab serial dilutions and pooled human
serum complement solution
Add CytoTox-GLO™ cytotoxicity reagent for quantifying the
cell death
Assay can measure between 50% to
156% relative CDC activity
CDC is an important mechanism of action for Adalimumab treatment of IBD
indication and hence a sensitive assay is important to characterize clinically
significant biological activity

39. Accelerate product development and reduce risk
39
1
2
3
4
5Early Product
Characterization
High resolution and
orthogonal approaches
provide enhanced
product understanding
In depth understanding
of the product enables
informed decisions
during product life
cycle
Due to the wide variety
of analysis methods
required, outsourcing
of PC activities may be
beneficial
Early stage adoption
of comprehensive
product
characterisation
reduces risk in
biologics
development
Use of partners with
the ability to provide
physico-chemical,
structural and
functional analysis can
simplify the PC process

40. BioReliance® Services product characterization facilities
40
Rockville, USA
High complexity physico-
chemical analysis
Mass Spectrometry
capabilities, including HDX
Binding analysis (SPR)
Cell based Assays
Stirling, UK
• Binding analysis (SPR
and other methods)
• Cell based assays
including CDC, ADCC
Glasgow, UK
• Physico-chemical
analysis including cIEF,
CE-SDS

41. BioReliance® Services
Analytical and bioanalytical testing
41
Services
Method transfer
or development
Method
validation
Reference
Characterization
(Innovator/
biosimilar)
Comparability
studies
Stability Testing
and Storage
GMP Lot
Release assays
Compendial
pH
Karl Fischer
Titration
Osmolarity
BCA & Bradford
Protein
Concentration
Appearance
SDS PAGE
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
Cell-based/
Immunoassays
Cell Based
Assays,
including
ADCC/CDC
Binding Assays
Kinetic Binding
Reporter
Bioassays

42. Principal Scientist, Global
Operational Development Services
BioReliance Services, Scotland
Phone: +44 (0) 1786 273077
Email:
pamela.hamill@bioreliance.com
Pamela Hamill, Phd
The vibrant M and BioReliance are trademarks of Merck KGaA, Darmstadt, Germany or its affiliates. All other trademarks are the property of their respective owners.
Detailed information on trademarks is available via publicly accessible resources.
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