Paving the way for Non-Clinical Bioanalytical Partnerships€¦ · “Many biotechnology-derived pharmaceuticals intended for human are immunogenic in animals. Therefore, measurement

Copyright © 2014 Covance. All Rights Reserved

Paving the way for

Non-Clinical

Bioanalytical

Partnerships

Louise Angell


Content

Overview of non-clinical immunogenicity testing

for biologics

Regulatory guidance

Bioanalytical considerations

Risk based approach

CRO Pharma


Overview of Immunogenicity Testing

All biologics (recombinant therapeutic proteins) will induce an immune response

Immunogenicity can be either wanted (vaccine) or

unwanted Binding (Ab binds to the drug but has no apparent impact of PK and

PD)

Sustaining (Ab binds to the drug and prolongs its half-life, increasing

drug exposure)

Clearing (Ab binds to the drug and accelerates clearance from

circulation, decreasing drug exposure)

Neutralizing (Ab binds to the drug and prevents pharmacological

activity)

Unwanted ADA response has

potential safety implications


Overview of Immunogenicity Testing

Test during non-clinical and clinical programs Immunogenic response in non-clinical study is not predictive of an

immune response in humans

Novel Therapeutic Protein May assume humanised protein to have less immunogenicity risk

than prokaryotic engineered protein

Humanised protein will have greater homology to native

sequence than non-humanised, however immunogenicity

frequency is still variable

Understand BioCMC and in vitro data

Assess in repeat dose non-clinical studies, multiple species to

enable interpretation of non-clinical data


Regulatory Guidelines - Summary

Regulatory agencies in the US and EU are consistent in their recommendation that immunogenicity be evaluated from a patient safety perspective due to non-predictability of data from non-clinical studies

Immunogenicity testing is required for novel compounds

Regulatory guidance for bioanalytical method validation is well defined for PK assays, with multiple white papers published for ADA assays, although ADA interpretation is evolving

For Biosimilar products, there is no global guidance for immunogenicity testing, and EMA infer in vivo testing is not required due to lack of predictability

in vivo studies may be considered reasonable markers for potential immunogenicity assessment due to differences in product specification (quality and manufacturing)


Regulatory Limitations

Potential for differing interpretation of the

guidelines by companies

Biosimilar (ADA Assays) Criteria for comparison of data?

It is not clear if one assay or two assay approach is optimal

Reference material?

One assay - additional assessments (cut point and drug tolerance?)

Pre-existing antibodies There is no regulatory guidance on how to deal with this


Bioanalytical Considerations


Overview of ADA assay

biotin-labeled drug

labelled drug

Streptavidin coated

plate

anti-drug antibody (ADA)


Overview of ADA assay

Qualitative screening assay, confirmation, titre quantification

Cut Point

-ve samples +ve samples

Assay signal

Re

spo

nse

Mean

5% False Positives


Insulin BioSimilar Case Study

EMA Guideline on non-clinical and clinical development of similar

biological medicinal products containing recombinant insulin and

insulin analogues (draft April 2014)

Risk based approach for toxicology studies

Presence of pre-existing antibodies against insulin is well

documented

biotin-labeled drug

Capture reagent

Streptavidin coated

plate

Anti-Drug Antibody

Anti-insulin antibody


Insulin Biosimilar Case Study Cut point set using naïve individuals however this included “positive” samples

Assay sensitivity was high (close to 1000ng/mL)

Some samples confirmed positive with both biosimilar and insulin

Potential to falsely report naïve background samples as confirmed positive

At risk of missing low concentration, true positives with high affinity binding

Many hours of Client communication to discuss concerns and risks

Rat sample analysis study: ~5% samples screened positive and no impact

upon TK profile

Learnings

Buffer cut point (sample with background greater than buffer would be

positive)

Pre-incubate cut point sample with insulin to remove pre-existing anti-

insulin Ab whilst capturing biological matrix variation

Skip confirmation step

Titre pre and post dose for comparison

Scientists came together to discuss potential issues


Non-Clinical Study Design

Some Clients have well defined BioCMC package and good

prediction of immunogenicity risk

Other Clients have limited experience with developing biologics

Dosing Human protein into mouse led to a high level of immunogenicity (60%)

Non-clinical program designed in 1 species

Immunogenicity response could not be correlated to the decrease in TK profile

because ADA samples were collected from different animals to TK samples

PD data subsequently generated to justify administration of human protein in

mouse model was responsible for apparent immunogenicity

Subsequently, ELISA method transferred and validation using Gyros to generate

ADA data from TK samples. This confirmed TK reduction correlated to ADA

response

These data were used to support absence of 2nd rodent model

Cyno study later demonstrated reduced/no immunogenicity

Enhanced relationships help to guide projects


Non-Clinical Study Design

Learnings for non-clinical sample collection and testing

Importance of pre-dose samples

Align ADA with TK sampling

Collect enough sample to allow tier testing (screen, confirmation, titer, nAb)

Design to account for sample volume limitations (rodents)

Choice of bioanalytical assay

Drug interference (extend wash-out period so drug is BLQ)


Bioanalytical Considerations

ADA In-Lab Analytical Time

Transfer 1-2 weeks

Development 2-3 weeks

Validation 3-4 weeks

Transfer Innovator + Biosimilar 1-2 weeks

Development Innovator + Biosimilar 2-3 weeks

Validation Innovator + Biosimilar 3-5 weeks

In 2014, ADA transfer studies lasted an average 6 week

in-life period

Not just analytical time, time is required for data

review, Client discussions, Client internal debate

What are Client (submission) timelines

Additional time for positive control generation


Improved Communication

Who is involved in a non-clinical drug development program?

Non-Clinical Program

Safety Assessment

Bioanalysis

Manufacturing

BioCMC

DMPK Client Study

Monitor

Discovery group

QA

Are

communications

well defined?


Risk Based Approach for ADA Testing


Interpretation of Immunogenicity

Immunogenicity must be considered with other endpoints

ADA…onset of immune response & change in PK

PK….pharmacokinetics (drug exposure)

PD….pharmacodynamics (drug activity)

AE….adverse events/observations (drug toxicity)

….all are important when interpreting study results!

ADA + PK + PD + AE = Immunogenicity Assessment

CRO challenge to generate data

Pharma challenge to consider all data and make

immunogenicity assessment

Pharma could increase efficiency of CRO by

ensuring data are shared


Risk Based Approach for ADA Testing Regulatory agencies consistently recommend that immunogenicity be

evaluated from a patient safety perspective

As such, companies should utilize a risk-based approach in evaluating

potential immunogenicity of their drug products

Immunogenicity is difficult to predict

– Potential immunogenicity of the protein

– Biological function of the protein

– Endogenous counterparts

– Route of administration

– Dose and frequency of administration

– Health status of subjects


Bioanalytical Cost Limitations

Is ADA testing required?

Is a fully validated method required?

Trastuzumab (Herceptin Biosimilar)

Supported several full validations (~3 weeks)

Assay sensitivity can be affected by drug tolerance

Cannot confirm ADA response in presence of several mg drug

Acid dissociation can affect sensitivity

Is it necessary to change non-clinical design?

Typical ADA sampling 0, 1, 4, 13 weeks

IgG t ½ = 6 weeks after 8 day response time

Costly to include wash out periods (TK trough samples)

Use risk based approach to understand BioCMC and in vitro data

Plan in vivo studies and consider commitment to ADA assay


Summary

Regulatory agencies in the US and EU are consistent in their

recommendation that immunogenicity be evaluated from a patient

safety perspective, however this is open to interpretation

CROs can be well placed to advise Clients of potential bioanalytical

limitations

Pre-existing antibodies

Non-clinical study design

Consideration to bioanalytical timelines

Increased communications between Pharma and CRO can benefit

Use risk based approach to consider extent of bioanalytical testing

required


Thank You


Additional info for reference as required


Regulatory Guidelines?

ICH ICH S6 Preclinical Safety Evaluation of Biotechnology-Derived

Pharmaceuticals (R1)

Q5E Comparability of Biotechnological/Biological Products Subject to

Changes in Their Manufacturing Process

Q2B Guideline Validation of Analytical Procedures Methodology

US Immunogenicity Assessment for Therapeutic Protein Products, August 2014

Scientific Considerations in Demonstrating Biosimilarity to a Reference

Product, February 2012 (draft)

Bioanalytical Method Validation, September 2013

23

“Many biotechnology-derived pharmaceuticals intended for human are immunogenic

in animals. Therefore, measurement of antibodies associated with administration of

these types of products should be performed when conducting repeated dose

toxicity studies in order to aid in the interpretation of these studies. Antibody

responses should be characterised (e.g. titer, number of responding animals,

neutralising or non-neutralising), and their appearance should be correlated with any

pharmacological and/or toxicological changes. …………“

“Animal immunogenicity assessments generally do not predict potential

immunogenic responses to protein products in humans. However, when differences

in manufacturing (e.g., impurities or excipients) between the proposed product and

the reference product may result in differences in immunogenicity, measurement of

anti-protein antibody responses in animals may provide useful information relevant

to patient safety.”


Regulatory Guidelines?

EU

Immunogenicity Assessment of Biotechnology-Derived Therapeutic

Proteins, 2007 (revision due)

Guideline on Similar biological medicinal products containing monoclonal

antibodies: non-clinical and clinical issues, 2010

Guideline on Similar biological medicinal products containing

biotechnology-derived proteins as active substance: non-clinical and clinical

issues , 2005

Guideline on Bioanalytical Method Validation (2011)

24

“……..the predictivity of non-clinical studies for evaluation of immunogenicity is considered low. Non-

clinical studies aiming at predicting immunogenicity in humans are normally not required. However,

ongoing consideration should be given to the use of emerging technologies …which might be used as

tools. Measurement of antibodies in non-clinical studies are however requested as part of repeated dose

toxicity studies, in order to aid in the interpretation of these studies ……….. the comparison of the antibody

response to the reference product in an animal model may be part of the comparability exercise both for

similar biological medicinal products ………. and for changes in manufacturing………

“Due to the different production processes used by the biosimilar and reference product manufacturers,

qualitative differences of process related impurities will occur……... Qualitative or quantitative difference(s)

of product-related variants ……may affect biological functions of the mAb and are expected to be

evaluated by appropriate in vitro assays. These quality differences may have an effect on immunogenic

potential and potential to cause hypersensitivity. It is acknowledged that these effects are difficult to predict

from animal studies and should be further assessed in clinical studies. Immunogenicity assessment in

animals is generally not predictive for immunogenicity in humans, but may be needed for interpretation of

in vivo studies in animals. Blood samples should be taken and stored for future evaluations if then

needed.”

“Non-clinical toxicity as determined in at least one repeat dose toxicity study, including

toxicokinetic measurements. ……………”


Immunogenic Classes of Therapeutic

Proteins

Class

Description Human

Protein

Homology

Immunogenicity

Frequency

Examples

A Prokaryotic Low High Staphylokinase

B Mammalian Low High OKT-3

C

Novel Construct Medium Variable High: Denileukin

Low: human

growth hormone

D Chimeric Human High Variable H: chMuL6

L: rituximab

E

Humanized High Variable L: Campath-1

H: Human anti-

CD3

F

Human

Identical

Variable H: GM-CSF

L: Human insulin

Bugleski and Treacy, Cur Opinion Mol Ther 6: 10-16, 2004

Strong

Weak


Animal Data Not Predictive

Protein Therapeutic Preclinical

Immunogenicity?

Clinical

Immunogenicity?

Streptokinase and

Staphlyokinase

High High

Keyhole Limpet Hemocyanin High in rodents High

Human interferon α- 2a High in rodents Low

Human Growth Hormone High in rodents Low

Human Interferon-λ High in Cynomolgus

monkeys

Low

Human Interleukin-3 High in Rhesus monkeys Low

Bugleski and Treacy, Cur Opinion Mol Ther 6: 10-16, 2004


Estimated Risk and Study Design Risk Level Drug Characteristics Examples Consequences

Low • Not structurally identical

to endogenous protein

• Non agonistic

• Enzymes

• Antibody drugs

• Infusion site reactions

• Loss of efficacy

• Mild allergic reactions

Medium • Partially or completely

identical to endogenous

protein

• Endogenous

counterpart is either

missing or redundant

or

• Not structurally


protein/agonistic

• Replacement therapy,

such as Factor VIII

• Antibody drugs

• Same as Low Risk

• Overstimulation of

endogenous mechanism

• Immune complex

formation

High • Partially or completely


protein

• Endogenous

counterpart is not

redundant

• Erythropoeitin

• GM-CSF

• Same as Medium Risk

• Neutralization of

endogenous counterpart


Estimated Risk and Study Design Bioanalytical Testing Strategy

Risk Level Sampling Frequency Assessment ADAs

Low More frequently earlier in

development, less frequently in

Phase III (baseline, end of study and

possibly follow-up)

Screen / Confirm

Titer assessment

Further characterization may be

helpful Nab, Mapping ADA

Medium More frequently earlier in

development, less frequently in

Phase III (baseline, end of study and

possibly follow-up)

Screen / Confirm

Titer assessment

Further characterization may be

helpful Nab, Mapping ADA

High More frequently throughout all

phases of clinical trails.

Consider real-time testing of ADA

and Nab

Screen / Confirm / Titer / NAb

Further characterization of ADA by

mapping, isotyping etc.

Sequential patient dosing rather

than cohort model for first-in-human

studies

Rosenberg and Worobec; Biopharm. Int. 17, 17, 18 (2004)

Documents

Paving the way for Non-Clinical Bioanalytical Partnerships€¦ · “Many biotechnology-derived pharmaceuticals intended for human are immunogenic in animals. Therefore, measurement