Analytical Development of Biosimilar Mabs: From Vision to Reality

SGS LIFE SCIENCE SERVICES WEBINAR

ANALYTICAL DEVELOPMENT OF BIOSIMILAR MABS: FROM VISION TO REALITYJune 25, 2014

Dr. Fiona M. GreerSGS Life Science Services

LIFE SCIENCE SERVICES OVERVIEWLIFE SCIENCE SERVICES OVERVIEW

1© SGS SA 2014 ALL RIGHTS RESERVED

SPEAKERSPEAKER

Dr. Fiona M. GreerGlobal Director for Biopharma Services D l tDevelopmentSGS Life Science Services


POLL QUESTIONPOLL QUESTION

What stage of development are you currently in with your Biosimilar?

(select all that apply)


ANALYTICAL AND CLINICAL DEVELOPMENT OF BIOSIMILAR mABs FROM VISION TO REALITY

MEETING REGULATORYMEETING REGULATORY CHARACTERIZATION EXPECTATIONSEXPECTATIONS

Dr Fiona M GreerDr Fiona M GreerGlobal Director, BioPharma Services DevelopmentSGS M-Scan

AGENDA: MEETING REGULATORY CHARACTERIZATION EXPECTATIONS

Introduction

What are the regulatory guidelines What are the regulatory guidelines associated with structural characterizationand comparability/biosimilarity testing of p y y gBiosimilars?

Wh i l ti l h t i ti i d? When is analytical characterization required?

Which techniques old & new are suitable Which techniques, old & new, are suitable Package of analytical tools/ battery of methods Strategies for primary and higher order structure


INTRODUCTION: REGULATORY PERSPECTIVE ON BIOSIMILAR MABS

June 2013, EMA approval of the first biosimilar mAbs in Europe (Celltrion’s Remsima™ and Hospira’s Inflectra™ versions of infliximab)

Many more biosimilar mAbs are currently in late-stage trials and can be expected to be submittedstage trials and can be expected to be submitted to Regulatory Authorities shortly

Jan 2014 Celltrion received S Korean MFDS Jan 2014, Celltrion received S. Korean MFDS approval for Herzuma, a version of Roche’s Herceptin trastuzumab

April 2014, Biocad received first approval of a biosimilar mAb in Russia for it’s rituximab, A llBi


AcellBia

GLOBAL REGULATORY BACKGROUNDGLOBAL REGULATORY BACKGROUND

EUROPE 2005, EMEA issued

guidelines on “Similar

UNITED STATES Biologics Price Competition and Innovation Act (BPCIA)

REST OF WORLD Brazil, Australia, Turkey, Taiwan Malaysiaguidelines on Similar

biological medicinal products”. Many updated since then.

( )March 23rd 2010. New pathway-351(k) in PHS Act.

Feb 2012, FDA issued draft Guidances (2 plus Q&A).

Taiwan, Malaysia, Argentina, Mexico, Japan, Canada, S.A. and others have some form of pathway.

Approved 1st Biosimilar in 2006 and now has 16 including 2 mAbs.

E i d l t

( p )

April 2013, draft Guidance on Formal Meetings Between the FDA and Biosimilar Biological product

p y

Some adopted EMA guides, others wrote their own.

Experienced regulatory authority.

Many non-European companies targeting this

g pSponsors or Applicants.

• May 2014, 5th draft Guidance.

Oct 2009, WHO “Guideline on Evaluation of Similar BiotherapeuticProducts”


companies targeting this market.

Products

EU REGULATIONS: ARTICLE 10(4) DIRECTIVE 2001/83/EC

Overarching Guideline (CHMP/437/04).“G id li Si il Bi l i l M di i l P d t ”Defines principles “Guideline on Similar Biological Medicinal Products”Defines principles

Quality

GeneralNon-

clinical

Clinical

General guidelines

Quality / Safety Efficacy

IFN-Epoetin LMWHGCSFSomatropinInsulin mAbs follitropin-a IFN-bpp

Product class specific data requirements

p

Non-clinical

Cli i l

Non-clinical

Cli i l

Non-clinical

Cli i l

Non-clinical

Cli i l

Non-clinical

Cli i l

Non-clinical

Cli i l

Non-clinical

Cli i l

Non-clinical

Cli i l

Non-clinical

Cli i l


requirements Clinical Clinical Clinical ClinicalClinicalClinical Clinical ClinicalClinical

US REGULATORY PATHWAY (1/2)US REGULATORY PATHWAY (1/2)

New pathway-351(k) requires comparison with a single reference product approved under the normal 351(a). Theapplication must include information demonstrating biosimilaritybased on data derived from:based on data derived from: Analytical studies demonstrating that the biological

product is “highly similar” to the reference product notwithstanding minor differences in clinicallynotwithstanding minor differences in clinically inactive components

Animal studies and A clinical study or studies A clinical study or studies

Two basic types of Biosimilar, from two steps: Biosimilar requires analytics (“highly similar”) Biosimilar - requires analytics ( highly similar ),

preclinical & clinical (including immunogenicity) studies, any of which can be waived

Interchangeable Biosimilar switching studies


Interchangeable Biosimilar - switching studies

US REGULATORY PATHWAY (2/2)US REGULATORY PATHWAY (2/2)

FDA will consider the “totality of the data and information submitted” Suggest the use of a “meaningful fingerprint-like analysis algorithm” May 13 2014, draft Guidance on “Clinical Pharmacology Data to Support a demonstration of Biosimilarity to a Reference Product”. Introduces 4 categories:

» Not similar» Similar» Highly similar» Highly similar» Highly similar with fingerprint-like similarity

Steven Kozlowski, M.D.Director, Office of Biotechnology ProductsOPS/CDER/US FDA


WHEN IS ANALYTICAL CHARACTERIZATION REQUIRED


REVISED EMA QUALITY GUIDELINE: COMPARABILITY EXERCISE

U t t f t l ti l th l th d Use state-of-art analytical, orthogonal methods

Comparative characterization studies should include assessment of composition physicalinclude assessment of composition, physical properties, primary and higher order structures, purity, product-related substances (e.g. isoforms) and impurities, and biological activity with “ ffi i tl iti l ti l t l ”

p g y“sufficiently sensitive analytical tools”

Quantitative ranges for quality attributes establishedestablished

Use material from final process for clinical trials (i.e. avoid additional comparability exercises)(i.e. avoid additional comparability exercises)

The suitability of the formulation should be demonstrated (need not be identical)


WHAT REGULATIONS COVER PHYSICOCHEMICAL CHARACTERIZATION?

ICH T i Q6B “S ifi ti T t ICH Topic Q6B “Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products”S l h i i d fi i Structural characterization and confirmation

1. Amino acid sequence2. Amino acid composition3. Terminal amino acid sequence3. Terminal amino acid sequence4. Peptide map5. Sulfhydryl group(s) and disulfide bridges6. Carbohydrate structure

Physicochemical properties1. Molecular weight or size2. Isoform pattern3. Extinction coefficient3. Extinction coefficient4. Electrophoretic pattern5. Liquid Chromatographic pattern6. Spectroscopic profiles


POTENTIAL ANALYTICAL TOOLSPOTENTIAL ANALYTICAL TOOLS

Amino acid sequence and modifications: MS, peptide mapping, chromatography

Glycosylation: Anion exchange, enzymatic digestion, peptide mapping, CE, MSmapping, CE, MS

Folding: MS S-S bridge determination, calorimetry, HDX and ion mobility MS, NMR, circular dichroism, Fourier transform spectroscopy, fluorescence

PEGylation & isomers: chromatography, peptide mapping Aggregation: Analytical ultracentrifugation, size-exclusion

chromatography, field flow fractionation, light scattering, microscopymicroscopy

Proteolysis: electrophoresis, chromatography, MS Impurities: proteomics, immunoassays, metal & solvents analysis S b it i t ti h t h i bilit MS Subunit interactions: chromatography, ion mobility MS Heterogeneity of size, charge, hydrophobicity:

Chromatography; gel & capillary electrophoresis, light scattering, IM-MS


CASE STUDY: ANTIBODY CHARACTERIZATION

Mass spectrometry of intact t i d l d L &H h iprotein and released L &H chains

Amino Acid Composition Analysis

N- and C-terminal sequencing Peptide “MAPPING” Analysis

(Sequence coverage: 100% LC and 100% HC)

Monosaccharide and sialic acid analysis

Oligosaccharide population analysis

SDS-PAGE analysis

Circular Dichroism

Analytical Ultracentrifugation


INTACT MASS MEASUREMENT

N-Linked biantennary core fucosylated with varying number of galactose residues

(MONITORING GLYCOSYLATION)

Fuc Man – GlcNAc

Asn - GlcNAc-GlcNAc- Man Man - GlcNAc

- Gal

- Gal

mAb +1 x G0F+ 1 x G1F

mAb +2 x G1F

G0F Mass shift = +1444 G1F Mass shift = +162G2F Mass shift = +324

+ 1 x G1F

mAb +2 x G0F

mAb +1 x G1F+ 1 x G2F


INTACT MASS COMPARISON OF THREE BIOSIMILAR MABS


PEPTIDE MAPPING WORKFLOWPEPTIDE MAPPING WORKFLOW


ANTIBODY ANALYSIS – GENERAL WORKFLOW


SULFHYDRYL GROUP(S) AND DISULFIDE BRIDGES


CHARACTERIZATION OF S S BRIDGESCHARACTERIZATION OF S-S BRIDGES

Disulphide bridged2567.0100

Voyager Spec #1 MC[BP = 3017.6, 2567]

Disulphide bridgedprotein

Enzymic/Chemicaldi ti

S S SH

Mixture ofpeptides

40

50

60

70

80

90

% In

tens

ity

KTCIVPEVSSVFIFPPKPK252 269

S S

SHE

E

digestion

2971.0 2989.6 3008.2 3026.8 3045.4 3064.0Mass (m/z)

00

10

20

30

40 C SS

KVTCVVVDISK280 289

SHE

EIdentification by MS

Followed by reductionA d f th MS

Reduction

And further MS1122.8

80

90

100

Voyager Spec #1=>SM5[BP = 1662.4, 7089]

754.3

80

90

100

Voyager Spec #1=>SM5[BP = 1662.4, 7089]1062.61988.1

VTCVVVDISK280 289

TCIVPEVSSVFIFPPKPK252 269

30

40

50

60

70

% In

tens

ity

30

40

50

60

70

% In

tens

ity


1154.0 1169.4 1184.8 1200.2 1215.6 1231.0Mass (m/z)

20

30

1955 1970 1985 2000 2015 2030Mass (m/z)

10

20

GLYCOSYLATION CHARACTERIZATIONGLYCOSYLATION CHARACTERIZATION

Structural characterisation and confirmation of carbohydrate.yFor glycoproteins, the following should be determined: Carbohydrate content (neutral sugars, amino sugars and sialic

acids)) Structure of the carbohydrate chains, the oligosaccharide pattern,

the antennary profile, linkage Glycosylation site(s) on the protein chain


ANALYSIS OF GLYCOSYLATIONANALYSIS OF GLYCOSYLATION

N-Glycans Intact Mass by MALDI orCOOH2HN

S---SS---S

N-GlycansO-Glycans

Intact Mass by MALDI or ES MSMonosaccharide Composition Analysis (LC & Reduction CarboxymethylationMS)

COOH2HNS-CM S-CMS-CMS-CM

Specific Protease DigestSpecific Protease Digest

PNG FPNGase F

Sep-pak Monosaccharide Composition

Reductiveelimination

0% 20% 40%

Permethylation MALDI,Nanospray MS/MS & Linkage analysis

CompositionGlycan Population ScreeningGlycan Antennary ProfileGlycosylation Site


Nanospray-MS/MS & Linkage analysisLC & MS methods

Linkage Analysis

GLYCOMIC / GLYCOPROTEOMIC WORKFLOW

Anomeric specificIntermonosaccharide Glycan

C iti

MALDI-MSLinkages Glycan Profile

HPAEC-PADHeterogeneity

& Extent of

Composition

Glycan

MALDI-MS

Native Glycans

Intact mass vs. Deglycosylated

ES MS / MALDI MS

Glycosylationy

Sequence

MALDI-MS/MS

A tSampleGlycoprotein

ES-MS / MALDI-MS

Quantitative Monosaccharide

Composition

DerivatisedGlycans

Antennary Profile

ESI-MS

GlycopeptidesComposition

GC-MS

Quantitative PMAA GC MS

Inter-monosaccharide

Linkages

Peptide mapping

Qualitative Site-specificGlycosylation

HPAEC-PAD

Quantitative Sialic AcidContent

PMAA GC-MS

Quantitative Glycan profile


LC-ES-MS2AB-LC-MS

y p

OLIGOSACCHARIDE PROFILINGLC- AND MS-BASED METHODS

2-AB labelling and HPLC-FLD for profiling Oligosaccharide population coupled with ESI-MS

TIC chromatogramAnnotations based on MS dataAnnotations based on MS data

25© SGS SA 2014 ALL RIGHTS RESERVED Example of IgG N-glycans

N-acetylglucosamineGalactose

MannoseFucose

N-acetylneuraminic acidN-glycolylneuraminic acid

CHARGE COMPARABILITYCHARGE COMPARABILITY

Imaging cIEF ofImaging cIEF of Monoclonal Antibodies


BIOPHYSICAL TECHNIQUES FOR HIGHER ORDER STRUCTURE CONFORMATION ANDORDER STRUCTURE, CONFORMATION AND AGGREGATION

Technique Reports on Advantages DisadvantagesTechnique Reports on Advantages DisadvantagesCircular Dichroism Secondary/ Tertiary

StructureQuantitativeSensitive to helix content Formulation buffers can interfere

QuantitativeFTIR Secondary Structure Sensitive to sheet content

Less prone to buffer interfence

IntrinsicFluorescence Local Tertiary Structure Sensitive

Potential for moderate HTP Qualitative

Extrinsic Fluorescence Surface hydrophobicitySensitiveEnsemble tertiary structure-no localPotential for moderate HTP

Qualitative

UV-VIS (2ndderivative) Local Tertiary StructureSimultaneous to concentration determination QualitativeUV VIS (2 derivative) Local Tertiary Structure determinationPotential for moderate HTP

Qualitative

Differential Scanning Calorimetry Thermal Stability

Screening method for formulation(HTP) Qualitative

SV-AUC Oligomers/ aggregates Matrix free, quantitative, resolution Slow,

DLS HMW aggregates Sensitivity, moderate for HTP Poor resolution, qualitative


SEC-MALS Oligomers/ aggregates Direct MW determination, rapid analysis Matrix presentHigh shear forces

SUMMARYSUMMARY

The development of a biosimilar requires comprehensive physicochemical structural characterization at many stages.

Initially, batches of originator are studied to determine the exact protein sequence PTMs and variability of quality attributesprotein sequence, PTMs and variability of quality attributes. These data form the Quality Target Product Profile (QTPP).

Advances in MS instrumentation and Proteomic/Glycomicstrategies enable rapid identification of QTPP including PTMsstrategies enable rapid identification of QTPP including PTMs.

At early stage, characterization surveys may help to guide choice of an appropriate cell line. Build similarity concept from start.

Various regulatory guidelines then require side-by-side comparative data to demonstrate “Biosimilarity”.

M ltiple orthogonal anal tical methods are sed to define Multiple orthogonal analytical methods are used to define “fingerprint” comparison. Strategies for primary and higher order structure determination are required.

Increasing importance on HOS to link with biological activity


Increasing importance on HOS to link with biological activity.

THANK YOU FOR YOUR ATTENTIONTHANK YOU FOR YOUR ATTENTION

Life Science Services Fiona GreerGlobal Director, BioPharma Services Development

SGS M Scan Ltd Phone: +44 (0) 118 989 6940+ 41 22 739 9548

SGS M-Scan Ltd Phone: +44 (0) 118 989 69402-3 Millars Business Centre, Fax: +44 (0) 118 989 6941Fishponds Close,Wokingham E-mail : [email protected], RG41 2TZ, UK Web : www.sgs.com/biosimilars

+ 1 866 SGS 5003+ 65 637 90 111

+ 33 1 53 78 18 79+ 1 877 677 2667

+ 33 1 41 24 87 87


+ 1 877 677 2667

Health & Medicine

Analytical Development of Biosimilar Mabs: From Vision to Reality