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
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SPEAKERSPEAKER
Dr. Fiona M. GreerGlobal Director for Biopharma Services D l tDevelopmentSGS Life Science Services
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POLL QUESTIONPOLL QUESTION
What stage of development are you currently in with your Biosimilar?
(select all that apply)
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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
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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
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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”
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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
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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
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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
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WHEN IS ANALYTICAL CHARACTERIZATION REQUIRED
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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)
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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
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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
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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
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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
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INTACT MASS COMPARISON OF THREE BIOSIMILAR MABS
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PEPTIDE MAPPING WORKFLOWPEPTIDE MAPPING WORKFLOW
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ANTIBODY ANALYSIS – GENERAL WORKFLOW
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SULFHYDRYL GROUP(S) AND DISULFIDE BRIDGES
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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
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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
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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
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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
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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
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N-acetylglucosamineGalactose
MannoseFucose
N-acetylneuraminic acidN-glycolylneuraminic acid
CHARGE COMPARABILITYCHARGE COMPARABILITY
Imaging cIEF ofImaging cIEF of Monoclonal Antibodies
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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
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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
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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
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+ 1 877 677 2667
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