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Critical aspects regarding the Critical aspects regarding the application of GLP Principles to application of GLP Principles to

new compounds such as new compounds such as biotechnology products biotechnology products

MariaMaria Mercede BrunettiMercede Brunetti

RTC RTC SpASpA ,, PomeziaPomezia , Rome, Italy, Rome, Italyee--mail:mail: mercebrmercebr @@rtcrtc .it.it

CoordinatorCoordinator --GLP group of GIQARGLP group of GIQAR

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INTRODUCTION

• Introduction

• Applicable GLP Principles & guidelines

• Conclusions - Final considerations

• Examples (cases)

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INTRODUCTION

Biopharmaceutical vs conventional products

• Biological & structural properties of product

� macromolecules (proteins, complexbiopolymers) vs small molecules (organic)

� biological/biotechnology-derived vsorganic synthesis

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• Study types: in vitro test /analytical methods

� Test & reference items� Analytical aspects

� characterisation� analysis of doses� analysis of biological matrixes

(TK/PK, immunogenicity)

Biopharmaceutical vs conventional products

INTRODUCTION

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GLP (OECD, 1997)

Applicable GLP Principles

GLP (Italy, 2007)

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OECD Advisory Document No.: 14, 2004

Applicable GUIDELINES (1)

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Applicable GUIDELINES (2)

ICH 6S, 1998

Farmacogenomics, 2005

Immunogenicity, 2008

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FDA - Bioanalytical method validation, 2001

Workshop - AAPS/FDA, 2007

Immunoassay, 2004

FDA Warning letters

Applicable GUIDELINES (3)

OECD OECD EventEvent , Villa , Villa TuscolanaTuscolana , Frascati (Roma), , Frascati (Roma), ItalyItaly , , AprilApril 10 10 –– 11,11, 2008 2008

OECD Advisory Document No.: 14, 2004

OECD OECD EventEvent , Villa , Villa TuscolanaTuscolana , Frascati (Roma), , Frascati (Roma), ItalyItaly , , AprilApril 10 10 –– 11,11, 2008 2008

PURPOSE: To facilitate the proper application and interpretation of the GLP Principles for the organisation and management of in vitro studies

• standard regulatory tests

• new methods/areas ( ...omics)

• high trough-put screening tests

OECD Advisory Document No.: 14

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APPLICATION

• Standard in vitro studies

OECD Advisory Document No.: 14

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• in vitro methods (part of in vivo studies)APPLICATION

OECD Advisory Document No.: 14

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EXAMPLES(cases)

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CASE - 1a

•Test item protein (vaccine, Ab)

•Type of study 13 wks in...

•Study Plan includes the evaluation of immunogenicity(in responsive specie)

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CASE - 1b

• REFERENCE ITEM ?

• ELISA TEST ?

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CASE-1c: reference item

Ab: Antibody/reference item

• Preparation• Purification• Quantitation and ...

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Ab: Preparation

• Must be prepared in a GLP study ?

CASE-1d: reference item

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Ab : Purification

GLP - characterisation & labelling:

• identity , concentration, storage condition , preparation date, expiry/retest date

CASE - 1e: reference item

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Ab: Quantitation and...

Ex.: in mg of protein/ml -Lowry

CASE-1f: reference item

GLP - characterisation & labelling:

• identity , concentration, storage condition , preparation date, expiry/retest date

Characterisation: meaning, extension ?

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FDA Guidance - Bioanalytical Method Validation (2001) - in house STD:• source and lot number, expiration date, certificate of analysis when available, and/or evidence of identity and purity

Characterisation: meaning, extension ?

GLP - characterisation & labelling:

• identity , concentration, storage condition , preparation date, expiry/retest date

CASE-1h: reference item

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CASE-1i: ELISA TEST

• set up• validation• analysis

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CASE-1l: ELISA TEST

SET UP ad hoc per studio

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CASE-1m: ELISA TEST

VALIDATION• Precision• Accuracy• LOQ (L/U) & range• Calibration curve• Matrix & buffer effects• Stability (freezing)

• acceptancecriteria

• reagents in house

• stability

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ANALYSES

• Storage ofsamples

CASE-1n: ELISA TEST

• acceptancecriteria

• reagents in house

• stability

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CASE-2: control matrix

� Characterisation ?

� Stability/retest date ?

� endogenous product ?

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CASE - 3a

• Test item: recombinant protein (therapeutic use )

• Type of study: repeated toxicity

• Study Plan includes:� evaluation of immunogenicity

(immuno-response present/absent )

� kinetic analysis (non-chromatographic methods)

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CASE - 3b

2 TYPES of TEST

• set up• validation• analysis

A: immunogenicity(test item + reference item - biosimilar)

B: kinetic (test item + reference item - biosimilar)

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CASE - 3c

☯ ELISA test in house ☯ Commercial Ab - surrogate

(mono or policlonal ?)

(antigen : test item in vivo study and/or reference item -biosimilar)

☯ Response: YES/NO (cut point definition)

A: immunogenicity - qualitative/semiquantitative(test item + reference item - biosimilar)

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CASE - 4a

• Test item DNA vaccine

• Type of studyrepeated toxicity

• Study Plan includes: biodistribution and evaluation ofimmunogenicity(in responsive strain)

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CASE - 4b

A: biodistribution: PCR(Sponsor - non GLP site in a multisite study-OK phase I)

B: immunogenicity humoral and cell-mediated (validation ? acceptance criteria ? scientific publications ?)

2 TYPE of TEST

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CASE - 4c

Semiquantitative analysis

☯ Humoral immunogenicity test� Reference item: commercial monoclonal Ab -

surrogate (calibration curve , QC)

� “ Test item ” : OGM (Balb 3T3/...antigen) - supplied by the Sponsor (through University)

Verification at receipt - acceptance (FACS- surrogate)

� Results analyses: criteria ?

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CASE - 4d

☯ cell-mediated immunogenicity test� Primary cells (splenocytes) conjugated or not with

peptide + fluorochrome (antigen - high and low concentration)

� Inoculum in treated mice: fluorescence measurement in samples (FACS)

� Results analyses: criteria ?

GLP: enough internal preliminary test/s ?

Semiquantitative analysis

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CASE - 5

� qualitative, quantitative andsemiquantitative analyses(PCR, FACS, ELISA, Multiplex, …)

Type of test: Biomarkers (exploratory)

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• SCIENTIFIC PERSONNEL (SD)

• PERSONNEL - Technicians

Human Factors: manual activities, variability

FINAL CONSIDERATION

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FINAL CONSIDERATION

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FINAL CONSIDERATION

RELATE WITH AUTHORITIESRELATE WITH AUTHORITIESRELATE WITH AUTHORITIESRELATE WITH AUTHORITIES

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THANKS to :

S. Cinelli & I. Andreini