In assessing the comparability of proposed biosimilar compounds to the innovator counterparts, regulatory agencies have stressed the "totality-of-evidence" approach, which relies on both structural and functional characterization, as well as data from animal and clinical studies. We present as a case study a package of assay methods developed for one such biosimilar, trastuzumab, which will include characterization of target binding, Fc receptor binding and ADCC activity as well as PK and immunogenicity assays to be used in clinical studies. Challenges encountered and approaches taken in the development of these methodologies are described..

1. www.eurofins.com
Product Characterization, PK and
Immunogenicity Assays for the
Development of Biosimilar Trastuzumab
John Kamerud, Ph.D.
Scientific Director
Eurofins Bioanalytical Services
Case Study

2. Eurofins Pharma
Bioanalytics Services US Inc
Eurofins Pharma Bioanalysis
Services UK Limited
 Pharmacokinetics
 Immunogenicity
 Biomarkers
 Biosimilar Testing
St Charles
Oxford

3. Breast Cancer

4. Breast Cancer
1 in 8 women will be diagnosed with Breast Cancer in
their lifetime
Breast Cancer is the second-leading cause of death
among women
Each year: 220,000 new cases in the US; 40,000 deaths

5. Breast Cancer Detection and Survival
Rates Are Improving
NCI SEER Stat Fact Sheets: Breast Cancer

6. Herceptin (Trastuzumab)
Herceptin® (Trastuzumab ) is a monoclonal antibody that selectively
binds with high affinity (kDa 5nM) to the human epidermal growth
factor receptor 2, HER2
HER2 is a transmembrane receptor tyrosine kinase
HER2 is overexpressed in 25-30% of breast cancers.
HER2 dimerizes with itself and other HER family members.
HER2/HER3 dimers stimulate downstream growth pathways
Overexpression of HER2 results in excess growth signals, resulting
in uncontrolled cell proliferation

7. Trastuzumab Mechanism of Action
Trastuzumab reduces the growth signal
• By binding to HER2 and preventing dimerization
Trastuzumab flags tumor cells for
ADCC
• By binding to HER2 and the FC receptors of
immune cells
Trastuzumab reduces levels of sHER2
• By binding to HER2 and preventing proteolysis

8. Herceptin (Trastuzumab)
Herceptin increases overall survival and reduces
probability of recurrence after surgery
Herceptin sales average around $6 billion per year
Patent expiry: 2014 (EU) 2019 (US)

9. Herceptin (Trastuzumab)
Herceptin increases overall survival and reduces
probability of recurrence after surgery
Herceptin sales average around $6 billion per year
Patent expiry: 2014 (EU) 2019 (US)
Trastuzumab Biosimilar Development

10. Herceptin Biosimilars in Development
Company name Product name Stage of development
Actavis/Amgen/Synthon,
USA/The Netherlands
ABP-980 Phase III trial expected to be completed in Dec 2016
Biocad, Russia BCD-022 Phase III trial expected to be completed in Nov 2014
Biocon/Mylan, India* CanMab ‘Similar biologic’ launched in India in Oct 2013
BioXpress Therapeutics,
Switzerland
- Biosimilar in pipeline
Celltrion, South Korea Herzuma Marketed in South Korea following approval in Jan
2014
Hanwha Chemical, South Korea HD201 Phase I study in Europe as of 2013
Hospira, USA CT-P6 Biosimilar licensed from Celltrion. Phase III trial in
Europe ongoing .
Oncobiologics/Viropro, USA - Biosimilar in development
Pfizer, USA PF-05280014 Phase I study completed. Phase III study planned
PlantForm, Canada - Clinical trials in humans expected to begin in 2014.
Stada Arzneimittel/Gedeon
Richter, Germany/Hungary
- Collaborating on biosimilars of trastuzumab and
infliximab
www.gabionline.net

11. Biosimilars: Regulatory Guidance
EMA Guidance on Similar Biological Medicinal Products
Containing Monoclonal Antibodies – Nonclinical and
Clinical Issues
Finalized May 2012
Guidance for Industry: Scientific Considerations in
Demonstrating Biosimilarity to a Reference Product
Draft issued February 2012
Guidance for Industry: Clinical Pharmacology Data to
Support a Demonstration of Biosimilarity to a Reference
Product.
Draft issued May 2014

12. Both Documents Outline a Stepwise Approach:
 Structural Characterization
 Functional Characterization
 Animal Studies
 Clinical Studies

13. Biosimilars: Stepwise Approach
Clinical
PK/PD
Preclinical
Biological
Characterization
Physicochemical
Characterization
Adapted from McCamish M and Woollett G. The state of the art in the development of biosimilars. 91(3):405–417)
Clinical
PK assay
ADA assay
Nab assay
Characterization
FcR & C1q
binding
HER2 kinetic
binding assay
ADCC assay

14. Required Assays
Functional
Characterization
In Vivo Studies
Target Binding Pharmacokinetics
Fc - receptor Binding Immunogenicity
Complement binding
Antibody Dependent
Cellular Cytotoxicity

15.  Standard approach is to measure kon and koff
directly.
 High flow rate so binding is not diffusion-limited
Target Binding
-200
0
200
400
600
800
1000
1200
0 200 400 600 800 1000 1200 1400 1600 1800 2000
Time s
Response
RU
kon koff

16. Challenge: Trastuzumab just won’t let go!

17.  HER2 immobilized to surface of a Biacore CM5 chip
 Trastuzumab at various concentrations flowed over
at a low flow rate (20 mL per minute)
 Response at equilibrium used in Scatchard
analysis to calculate “psuedo” Kd
 Also can use response curves to compare potency
of different preparations
Target Binding

18. Scatchard plot
y = -0.0354x + 26.771
R² = 0.9872
-5
0
5
10
15
20
25
30
0 100 200 300 400 500 600 700 800 900
Response(RU)/Conc(nM)
Response (RU)

19. Potency comparison
0
100
200
300
400
500
600
700
800
900
1000
0.0 0.1 1.0 10.0 100.0 1000.0
Response (RU)
Trastuzumab (µg/mL)
Ref= Lot: 100%
Reference
Uk1= Lot: 130%
Reference
Uk2= Lot: 80%
Reference
Uk3= Lot: 120%
Reference

20. Trastuzumab
Potency Assay Qualification
Qualification
Principle
Qualification
Parameters
Acceptance
Criteria
Qualification
Results
Assay Linearity
and Range
Correlation coefficient
Y-intercept
Slope
Residual sum of squares
> 0.95
-15% to +15%
0.8 to 1.2
report result
0.995
-3.66
1.05
31
Repeatability
(Intra-assay precision)
%CV <20% <7%
Intermediate
precision
(Inter-assay precision)
%CV <20% <4%
Accuracy Recovery 80% to 120% 99.9% to 102.9%

21.  FcγRI – CD64
 FcγRII – CD32a
 FcγRIII – CD16a
 FcRn
Fc Receptor Binding

22. Receptor
name
Principal
antibody
ligand
Affinity for
ligand
Cell
distribution
Effects of
binding to
antibody
FcγRI (CD64) IgG1 and IgG3 High (Kd ~ 10−9 M)
Macrophages
Neutrophils
Eosinophils
Dendritic cells
Phagocytosis
Cell activation
FcγRIIA (CD32) IgG Low (Kd > 10−7 M)
Macrophages
Neutrophils
Eosinophils
Platelets
Langerhans
Phagocytosis
Degranulation
FcγRIIIA
(CD16a)
IgG Low (Kd > 10−6 M)
NK cells
Macrophages
ADCC
cytokine release
FcRn IgG
Epithelial cells
Endothelial cells
Hepatocytes
Maternal / fetal
transfer
Protects IgG
from degradation
Fc Receptor Binding

23. Trastuzumab: CD16a binding
0.0
200.0
400.0
600.0
800.0
1000.0
1200.0
1400.0
1600.0
1800.0
1 10 100 1000 10000
Response (RU)
Trastuzumab (mg/mL)
Reference
70%
100%
130%

24. Trastuzumab
CD16a Binding Assay Qualification
Qualification
Principle
Qualification
Parameters
Acceptance
Criteria
Qualification
Results
Assay Linearity
and Range
Correlation coefficient
Y-intercept
Slope
Residual sum of squares
Range
>0.95
Report result
0.8 to 1.2
Report result
Report result
0.99
-4.90
1.0
54.9
7.8 µg/mL to 1000 µg/mL
Repeatability
(Intra-assay precision)
%CV
Mean
Range
1.5%
0.1 – 10.6%
Intermediate
precision
(Inter-assay precision)
%CV
Mean
Range
5.0%
1.1 – 13.1%
Accuracy Recovery 80% to 120% 93.7 – 102.6%

25. Complement Component C1q
First component of complement cascade
Binds to IgG or IgM which is complexed to target antigen
Low affinity for single IgG Fc
Difficult to measure using SPR
ELISA developed to measure binding of C1q to immobilized MAb

26. HRP anti-C1q antibody
Complement C1q binding
Coat drug at multiple
concentrations
Human C1q

27. 0.000
0.500
1.000
1.500
2.000
2.500
3.000
3.500
4.000
1 10
OD
Trastuzumab(µg/mL)
S (Reference)
Uk1 (70%)
Uk2 (100%)
Uk3 (130%)
Trastuzumab - C1q binding

28. Antibody-dependent Cellular Cytotoxicity
Major mechanism of action for oncolytic Mabs
Several methods for measuring ADCC have been developed:
- Release of radioactive chromium
- Release of LDH
- Activation of engineered effector cells
Our approach: cytotoxicity of target cells using Flow Cytometry

29. ADCC
Flow Cytometry
CFSE
Step 1
Step 2
Step 3
+ +
+
FACS
+

30. ADCC - Trastuzumab
0.0
500.0
1000.0
1500.0
2000.0
2500.0
3000.0
0.10 1.00 10.00 100.00 1000.00
Normalizedlivetargetnumber
Herceptin (ng/mL)
S (Reference)
Uk1 (50%)
Uk2 (100%)
Uk3 (150%)

31.  Comparative toxicology study, including TK
 PK / PD (maybe)
 Immunogenicity (interpretation of TK)
Animal Studies
Clinical Studies
 Bioequivalence – head-to-head
 PK / PD expected
 Immunogenicity

32.  FDA and EMA do not define acceptance criteria
for comparability, but state this should be
defined a priori on a case by case basis
 However…..
 MOST biosimilars approved to date have used
the standard bioequivalence criteria:
 The 90% CI of the ratio of mean PK parameters
(AUC, eg) should be within 0.8 – 1.25
PK Studies:
Assessing Biosimilarity

33.  As with any bioequivalence study, minimizing
analytical differences is imperative, as is
minimizing variability
 One assay method for both products
 Demonstrate equal reactivity in development
 Ideally, same calibrator, separate QCs for
innovator and biosimilar drugs
 Good precision and selectivity will help reduce
variability
PK Studies:
Considerations for Assay Development

34. Consensus around one-assay strategy

35.  No between-assay variability; ie, minimization of the
potential impact of assay bias on the comparison of the
biosimilar and reference product
 Blinded study sample analysis possible
 Need to develop and validate only one assay*
*Conservative approach is to use biosimilar curve for
quantitation of both biosimilar and reference drug
concentrations
Advantages of one-assay strategy

36.  Compare curves in development
 Establish in validation using QCs
 Both Reference and Biosimilar QCs should meet
standard acceptance criteria:
RE < 20% (25% at LLOQ and ULOQ)
CV < 20% (25% at LLOQ and ULOQ)
 Absolute difference between RE(ref ) and RE(biosim ) should
be < 20% (25% at LLOQ and ULOQ)
 90% CI of the difference should be < 30% (35% at LLOQ
and ULOQ)
Recommended approach to assess
bioanalytical similarity

37. “A true comparison of immunogenicity across
different products in the same class can best
be obtained by conducting head-to-head
patient trials using a standardized assay that
has equivalent sensitivity and specificity for
both products.”
FDA Draft Guidance on Immunogenicity (2009)
“The proposed product and reference product
should be assessed in the same assay with
the same patient sera whenever possible.”
FDA Draft Guidance on Biosimilars (2012)
Anti-drug Antibody: One Assay or Two?

38. Anti-drug Antibody: One Assay or Two?
Consensus is forming around One-Assay strategy for
ADA.
Labeled reagents should be made from biosimilar, not
innovator.
There is a slight risk that some antibodies to reference
product won’t be detected.

39. Challenges for PK and ADA assays
PK: Potential interference from shed receptor (HER2):
• Soluble HER2 levels may be as high as 1880 ng/mL
(median = 11 ng/mL)
• < 500 ng/mL in 94% of patients
ADA: Drug interference:
• Predicted concentrations are roughly 50 mg/mL
(trough) to 150 mg/mL (peak)

40. PK assay
Format
Shed receptor
Trastuzumab
Acid dissociate
Neutralize
Trastuzumab
HER2 extracellular domain
Biotinylated anti-ID

41. PK assay: Performance results
Performance characteristic Results
Validated Range
(LLOQ/ULOQ) 1.5 µg/mL to 80 µg/mL
Accuracy Human Serum
Range 3.3% to 9.3%
Mouse Serum
Range 3.8% to 9.1%
Precision
Intra-assay
Inter-assay
Human Serum
Range 5.0 % to 16.1 %
Range 7.3 % to 16.6 %
Intra-assay
Inter-assay
Mouse Serum
Range 2.6% to 9.0 %
Range 6.8 % to 13.2 %
Specificity / Selectivity 10 out of 10 lots of human serum within ±20% of nominal
(levels tested for each lot: unspiked, 10 and 50 µg/mL)
Dilutional Linearity %RE Range: -8.5 % to -22.1 %; Overall %CV: 7.5 % Maximum
dilution performed 1:50 (exclusive of MRD)
Soluble Target Interference
(sHER2)
No interference observed up to 4000 ng/mL sHER2

42. OH-
H+
Free drug Anti-drug antibody
1: Acid dissociate and
neutralize
2: Capture on drug-coated
plate
ADA
“ACE” assay format (1)

43. Coated Plate Uncoated Plate
3. Elute using acid
4. Neutralize and
coat on second
plate
5. Detect with
biotinylated drug
and SA-HRP
H+
Biotinylated drug
Streptavidin-HRP
ADA
“ACE” assay format (2)

44. ADA assay: Performance results
Performance
characteristic
Results
Sensitivity 5 ng/mL
Cut Point Assessment 30 individual female sera samples
Floating cut point factor was established at 1.32
Selectivity (Matrix
recovery)
12 out of 12 lots of human serum lots spiked with
50 ng/mL ADA were within ±25% of reference
Precision
Intra-assay
Inter-assay
3.4%
19.6%
Drug Tolerance 500 ng/mL anti-trastuzumab ADA is detectable
in the presence of 62.5 µg/mL of trastuzumab

45. Conclusions
• Evaluation of the comparability of trastuzumab biosimilars to the
innovator drug should follow the guidelines laid out by the FDA
and EMA.
• The analysis should be multifactorial, taking into account both the
physicochemical characteristics and clinical performance of the
biosimilar compared to the innovator.
• Each compound is unique and each method poses unique
challenges.
• Facilitating the availability of biosimilar trastuzumab will provide
additional options for breast cancer patients.

46. Biosimilar Analysis At Eurofins
Eurofins Bioanalytical Services offers a full range of
pre-qualified assays for comparability testing of
biosimilars for development (exploratory and GLP)
Characterization
FcR & C1q
binding
HER2 kinetic
binding assay
ADCC assay
Clinical
PK assay ADA assay Nab assay
Trastuzumab Bevacizumab Cetuximab Rituxumab Insulins
www.eurofins.com/bioanalyticalservices

47. Thank You!
Acknowledgements
Matt Bentley
David Shaw
Zhe Liu
Diane Werth
Craig Draper

48. Q&A
JohnKamerud@Eurofins.com