1. Immunogenicity testing of biologic products relies entirely on the design and execution of the assays used, making it impossible to directly compare reported immunogenicity rates between products unless a head-to-head clinical study is conducted. 2. Multiple factors influence reported immunogenicity rates, including the assay format, sensitivity, controls, and clinical study design. The choice of assay cut-off also impacts the rate of samples deemed positive or negative. 3. Even in head-to-head clinical studies, biosimilar companies may report only relative immunogenicity compared to the reference product due to differences in assay methodology and validation between companies. Direct comparison of rates is generally not possible without identical assay methodology.

1. “Biotherapeutic Medicines – regulatory challenges and current
practices. Approaches for harmonization” Moscow, Russia May 16,
2013. IFPMA/AIPM
Immunogenicity testing of Biotechnology
Products and the Impact to Biosimilars
Barry Cherney
Executive Director Product Quality
Amgen Inc

2. 2
• Administration of non native or even ‘humanized’
proteins to either animals or humans can elicit
either an antibody response, cellular response, or
both if the immune system recognizes the protein
as foreign.
• All protein products have some level of
immunogenicity, with varying impact to patients.
• Although there are multiple theoretical causes of
immunogenicity, very few real examples of why a
product is immunogenic have been published
Protein Products have Immunogenic
Potential

3. 3
Generalized Protein Domains
Receptor
binding/functional domain
Non-receptor
binding/functional domain

4. 4
Effect of antibodies on the function of
proteins
Cell Response Neutralizing
antibodies
Non-neutralizing
antibodies
No cell
response

5. 5
Clinical Concerns in Testing for
Antibodies - Effects on PK/PD
• The binding of antibodies to product has been
shown to potentially affect (by extension or
reduction) the half life in blood through the
influence on clearance mechanisms.
• Biodistribution of product has also been
shown to be affected, such as lack of
targeting to skin or tumour sites.
• If product is cleared differently and its
distribution is different, then its ability to have
the desired biological effect may also be
altered.

6. 6
Clinical Concerns in Testing for
Antibodies - Effects on Efficacy
• Antibodies that alter the PK/PD of the
product may have an effect on its efficacy. If
the drug remains longer in the circulation,
efficacy can be enhanced. If half life is
reduced, so potentially, is its efficacy.
• The presence of neutralizing antibodies can
directly inhibit the biological activity of the
product and thus clinical efficacy may be
reduced or abrogated.

7. 7
Clinical Concerns in Testing for
Antibodies - Effects on Safety
• Extending the half life of a product can
influence its toxic properties.
• Redistributing a product to different sites may
potentially also have safety implications.
• The presence of complexes of product and
antibody can have physiological
consequences.

8. 8
• Immune responses to product can lead to:
• Anaphylaxis
• Injection site reactions
• Flu like syndromes
• Allergic responses
• One of the most serious adverse events
occurs when neutralizing antibodies to
product cross react with endogenous proteins
that have a unique physiological role.
Clinical Concerns in Testing for
Antibodies - Effects on Safety

9. • “Comparative assessment of unwanted immune responses against
the biosimilar and the reference mAb are normally undertaken as
part of the clinical study”*
• “A risk-based approach can provide a starting point from which the
further concept of immunogenicity testing can be designed, but due
to the diversity of risk factors, as discussed in this guideline, and the
variety of mAbs and mAb-related products, the recommendations
given here cannot be generalized.”**
• “Assessment is based on the identification of risk factors inherent to
the particular mAb in question, the final drug product and the treated
patient population. The mechanism of action and the basic structure
(chimaeric, humanized, fully human) are not sufficient for deciding
on the attribution of risk level. For a risk-based approach, applicants
need to define what “risk” in this context means.”**
* Guideline on similar biological medicinal products containing monoclonal antibodies – non clinical
and clinical issues CHMP 2010 ** Guideline on immunogenicity assessment of monoclonal
antibodies intended for in vivo clinical use. CHMP Nov. 2010
EMA mAb biosimilar and immunogenicity
guidelines on the “risk-based approach”
9

10. 10
• “At least one clinical study that includes a
comparison of the immunogenicity of the proposed
product to that of the reference product will
generally be expected “
• “The extent and timing …..of the clinical
immunogenicity program will vary depending on a
range of factors including the extent of analytical
similarity and the incidence and clinical
consequences of immune response for the
reference product. “
*Scientific Considerations in Demonstrating Biosimilarity to a Reference Product
FDA. Feb 2012
FDA’s draft biosimilar guideline* also
includes “a risk based immunogenicity
approach”

11. Risk Based Approaches to
Immunogenicity Testing
Risk = Probabilityharm x Severityharm
• Severity outweighs the probability of a risk
occurring.
How many patients are
likely to mount an immune
response?
What happens to the patient
if they mount an immune
response?
11

12. Considerations in Assessing Risk of
Immunogenicity - Probability Analysis
Likely Lower Probability Likely Greater Probability
Immunosupressed patients Autoimmune disease
Single dosing Chronic dosing
More ‘Human’ ‘Foreign’
IV administration Subcutaneous
Highly pure Impure
No aggregates Aggregates
12
Risk = Probabilityharm x Severityharm

13. 13
Considerations in Assessing Risk of
Immunogenicity - Severity Analysis
Likely More Severe Likely Less Severe
Endogenous version No endogenous version
Unique activity Redundant activity
Sole therapy Other therapies
Life threatening disease Non life threatening disease
Chronic disease End stage disease
Non reversible AE Reversible AE
Replacement therapy Non replacement therapy
Anaphylactic response Non anaphylactic response
Risk = Probabilityharm x Severityharm

14. Examples of Anti Drug Antibodies: Incidence
and Clinical Impact
14
Rate of
Antibodies
Molecule
Antibody
Incidence
Clinical Impact
High
r-Human alpha-
galactosidase
88% none reported
High
r-chimeric
anti-TNFa
10-57%
Hypersensitivity
PK affected
efficacy unchanged
Moderate
r-chimeric
anti-GPIIb/IIIa
Fab
7-19%
Higher incidence after
re-administration,
TCP higher in ab+
patients
Moderate
r-Human
Glucocere-brosidase
13%
Neutralizing
antibodies
rare
Low
r-Human
Thrombopoietin
truncated, PEG
0.5-4%
neutralizing
antibodies
TCP
Low
r-Human Tissue
Plasminogen
Activator
<1% none reported

15. Considerations for Analytical Testing Strategies based on the
outcome of the Risk Assessment Score
Examples of High score
considerations
Examples of Low score
considerations
Test samples in real time Batch test samples at the end of
the study
More sensitive assays early in
development
Develop increasingly sensitive
assays during development
Highly conservative approach
to setting cut points
Less conservative approach to
setting cut points
Test positive binding samples
for neutralization in real time
Batch positive binding samples
after initial screening and then
test for neutralization
Need to develop sensitive
neutralization assays
Less need for very sensitive
neutralization assays
15

16. 16
Biosimilar Specific Issues with
Immunogenicity Testing
• The rate of immunogenicity detected in an assay is wholly
dependent on the assay chosen, its controls, execution
and the clinical study sample plan design
• Biosimilar companies are unlikely to know the details of
the innovator assays:
• Assay format (RIA, ELISA - bridging or direct etc.)
• Assay sensitivity and specificity
• Assay positive control (standard)
• Positivity criteria
• Timing and number of samples
• Patient population
• Multiple indications
(if going into licensure with only one)
Google.www

17. The Optimal Screening Assay
An Optimal screening assay should:
• Be sensitive enough to detect any level of
specific antibody present in patient sera.
• Have no false negatives
• Have a few false positives able to be
proved false positives
• Discriminate between pre-existing
antibodies and treatment induced
antibodies
• Detect antibodies in the presence of drug
• Be reproducible
17

18. 18
The Choice of Assay Format can Heavily Influence
the Rate of Immunogenicity Results Reported
Enzyme
Coloured
reagent
Clear
reagent
Detecting
antibody
Anti-drug
antibody
Drug
Serum
Antibody
Conjugated
Drug
Drug
Detection
System
Add Serum
Sample
Precipitate antibody
bound drug
Measure
radioactivity
in pellet
ELISA
Bridging
ELISA
Radio
Immunoprecipitation
Assay (RIP)

19. 19
Each Assay Format Has Implications for the
Results it Reports
• ELISAs do have several issues related to design,
especially non specific background and the need to
stick the antigen to a solid surface
• The extensive washes required to reduce background
can reduce the ability to detect low affinity antibodies
as they can get washed away
• Incubation times may not allow for sufficient binding of
low affinity antibodies- too long can lead to
dissociation too

20. Each Assay Format Has Implications for
the Results it Reports
• The bridging format appears to have the least
background and does not require the use of an anti-
human Ig reagent and its associated validation. Care
must be taken to avoid loss of low affinity antibodies
due to the bispecific nature of the binding.
• RIP assays tend to be the more sensitive assay
format as compared to ELISAs*. Not all methods to
precipitate complexes, such as Protein A/G,
necessarily detect all Ig isotypes and subclasses and
the format of choice should be justified by the sponsor
*Swanson, S.J., et. al., (2004) Nephron Clin Practice Vol 96 p88-95
20

21. 21
Surface Plasmon Resonance (SPR)

22. 22
SPR Assays - Advantages and
Disadvantages
• SPR assays involve the exposure of drug in a lawn of
dextran that do not affect the structure of the product
to the extent of an ELISA
• It is a real-time procedure and is therefore fast and
also detects rapidly dissociating antibodies which can
be missed by other methods
• SPR assays do not require extensive wash steps and
can not only detect low affinity antibodies but
characterize the binding activity
• SPR equipment is expensive and needs expert
knowledge to design, run and analyze data if the
results are not to be spurious

23. 23
Selecting the Assay Cut Point Impacts
Reporting Rates
0.000
0.500 1
5
9
13
17
21
25
29
Sample Number
Mean
NSB
Cutpoint
at 95%
negative
X
OpticalDensity(OD)
Negative
Control
• How the assay cut point is selected dictates when a
sample is deemed negative or positive
• The assay cut point thus has impact on the rate of
seroconversion reported

24. 24
Circulating Therapeutic Can Interfere With
Antibody Detection in Antibody Assays
Drug Coat
Circulating
Drug
Serum Anti
Drug Antibody
No Binding to Plate
Drug interference can cause significant problems in detection of antibody responses due to
the presence of product in samples collected for antibody assessment. This normally
results in an artefactually low estimate of antibody content of affected samples and can be
so pronounced as to cause false negative results

25. One has to Understand the Impact of Drug in the
Sample if the Screening Assay is to be Meaningful
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50 0
0.08
0.16
0.32
0.63
1.25
2.5
5.0
10
20
40
80
160
320
640
1280
µg/ml drug added to Serum Sample
Titer 3.1
Titer 2.9
Titer 2.7
Titer 2.5
Limit of
detection
Titre
25

26. 26
Identification of Immunoglobulin Class
• It may be useful to identify the class and subclass of
immunoglobulin detected in a sample but depends
on the product and patient responses:
• The disease state or route of administration of
drug induces an antibody response of classes
other than IgG
• IgA in certain skin autoimmune disorders
• IgE often manifests itself in the patient before
being detected in a screening assay.
• Should hypersensitivity be detected in clinical trials,
the development of an assay is warranted
• This may be able to detect and avoid reactions to
subsequent doses

27. A Histamine Release Assay to Measure
Anti-drug IgE
IgE receptor
transfected cell
IgE
IgE loaded cell
Drug
IgE receptors
crosslink
Histamine
Release
27

28. Neutralizing Capacity of Antibodies
•The neutralizing capacity of antibodies to many products
should be tested in a biological assay
•Alternative binding-based assays may be appropriate for
certain products particularly when used as the potency
assay for release– which are often more sensitive than cell
based assays
•There is discussion about the utility of neutralizing
antibody assays in respect to clinical impact (in vitro tests
may not reflect what occurs in vivo).
•From a risk based approach, identification of a neutralizing
response can allow for subset analysis of weak signals in the
patient population – patients with a neutralizing response may be
assessed for PK and PD but this may not be straightforward (e.g. in
the oncology setting)
28

29. 29
Bioassays for Neutralizing Antibodies
• Assay responses to
product may take
the form of cell
proliferation or
growth inhibition,
secretion of protein,
gene expression
etc.
Cell Response
No Cell
Response
Product
Neutralizing Antibody

30. 30
A Cytokine Neutralizing Antibody Bioassay
0.000
0.200
0.400
0.600
0.800
1.000
1.200
1.400
1.600
1.800
2.000
1 10 100 1000 10000 100000
Abs450
Serum Concentration [Dilution]
Cytokine
Serum Pre Exposure
Serum Post Exposure

31. 31
Conclusions - Immunogenicity
1. The results of immunogenicity testing rely wholly on the design
and execution of the assay used to detect and characterize
any immune response
2. It is impossible to compare rates of immunogenicity between
biotechnology products unless a head to head clinical
immunogenicity study is carried out, thus they should be
conducted as such
3. Even in such head to head studies, the rates of
immunogenicity are dependent on the assay that the biosimilar
company uses and may not reflect the rates seen by the
innovator, thus only ‘relative’ assessments of rates can be
provided.

32. Acknowledgements
• Tony Mire-Sluis
• Gino Grampp
• Andrew Fox
• Geoff Eich
• Richard Markus
32