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

Eurofins Pharma

Bioanalytics Services US Inc

Eurofins Pharma Bioanalysis

Services UK Limited

Pharmacokinetics

Immunogenicity

Biomarkers

Biosimilar Testing

St Charles

Oxford

Breast Cancer

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

Breast Cancer Detection and Survival

Rates Are Improving

NCI SEER Stat Fact Sheets: Breast Cancer

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

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

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)

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

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

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

Both Documents Outline a Stepwise Approach:

Structural Characterization

Functional Characterization

Animal Studies

Clinical Studies

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)

Clin

ical PK assay

ADA assay

Nab assay

Ch

ara

cte

riza

tion

FcR & C1q binding

HER2 kinetic binding assay

ADCC assay

Required Assays

Functional

Characterization

In Vivo Studies

Target Binding Pharmacokinetics

Fc - receptor Binding Immunogenicity

Complement binding

Antibody Dependent

Cellular Cytotoxicity

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

Tim e s

Re

spo

ns

e

RU

kon koff

Challenge: Trastuzumab just won’t let go!

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

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

Resp

on

se (

RU

)/C

on

c (

nM

)

Response (RU)

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

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%

FcγRI – CD64

FcγRII – CD32a

FcγRIII – CD16a

FcRn

Fc Receptor Binding

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

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%

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%

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

HRP anti-C1q antibody

Complement C1q binding

Coat drug at multiple

concentrations

Human C1q

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

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

ADCC Flow Cytometry

CFSE

Step 1

Step 2

Step 3

+ +

+

FACS +

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

No

rma

lize

d liv

e t

arg

et

nu

mb

er

Herceptin (ng/mL)

S (Reference)

Uk1 (50%)

Uk2 (100%)

Uk3 (150%)

Comparative toxicology study, including TK

PK / PD (maybe)

Immunogenicity (interpretation of TK)

Animal Studies

Clinical Studies

Bioequivalence – head-to-head

PK / PD expected

Immunogenicity

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

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

Consensus around one-assay strategy

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

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

“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?

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.

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)

PK assay Format

Shed receptor

Trastuzumab

Acid dissociate

Neutralize Trastuzumab

HER2 extracellular domain

Biotinylated anti-ID

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

OH-

H+

Free drug

Anti-drug antibody

1: Acid dissociate and neutralize

2: Capture on drug-coated plate

ADA “ACE” assay format (1)

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)

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

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.

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

Thank You!

Acknowledgements

Matt Bentley

David Shaw

Zhe Liu

Diane Werth

Craig Draper

Q&A JohnKamerud@Eurofins.com