The approval of the first biosimilar mAbs in Europe last June (Celltrion’s Remsima™ and Hospira’s Inflectra™ versions of infliximab) paves the way for the advent of more widespread access to biological treatments in indications such as rheumatology and oncology. One year on, lessons learned from these approvals will guide the global regulatory framework for such complex biomolecules. Indeed, several more biosimilar mAbs are currently in late-stage clinical trials and can be expected to be submitted to Regulatory Authorities shortly. Read more or listen to live streaming http://bit.ly/SGSBiosimilarWebinar

1. SGS LIFE SCIENCE SERVICES WEBINAR
ANALYTICAL DEVELOPMENT OF
BIOSIMILAR MABS: FROM VISION
TO REALITY
June 25, 2014
Dr. Fiona M. Greer
SGS Life Science Services

2. LIFE SCIENCE SERVICES OVERVIEWLIFE SCIENCE SERVICES OVERVIEW
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3. SPEAKERSPEAKER
 Dr. Fiona M. Greer
Global Director for Biopharma Services
D l tDevelopment
SGS Life Science Services
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4. POLL QUESTIONPOLL QUESTION
 What stage of development are you currently
in with your Biosimilar?
(select all that apply)
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5. ANALYTICAL AND CLINICAL DEVELOPMENT OF BIOSIMILAR mABs FROM
VISION TO REALITY
MEETING REGULATORYMEETING REGULATORY
CHARACTERIZATION
EXPECTATIONSEXPECTATIONS
Dr Fiona M GreerDr Fiona M Greer
Global Director, BioPharma Services Development
SGS M-Scan

6. AGENDA: MEETING REGULATORY
CHARACTERIZATION EXPECTATIONS
 Introduction
 What are the regulatory guidelines What are the regulatory guidelines
associated with structural characterization
and comparability/biosimilarity testing ofp y y g
Biosimilars?
 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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7. 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

8. 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 p
Sponsors or Applicants.
• May 2014, 5th draft
Guidance.
 Oct 2009, WHO
“Guideline on Evaluation
of Similar Biotherapeutic
Products”
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companies targeting this
market.
Products

9. 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
General
Non-
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
8© SGS SA 2014 ALL RIGHTS RESERVED
requirements Clinical Clinical Clinical ClinicalClinicalClinical Clinical ClinicalClinical

10. 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). The
application must include information demonstrating biosimilarity
based 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

11. 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 Products
OPS/CDER/US FDA
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12. WHEN IS ANALYTICAL
CHARACTERIZATION REQUIRED
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13. 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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14. 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 sequence
2. Amino acid composition
3. Terminal amino acid sequence3. Terminal amino acid sequence
4. Peptide map
5. Sulfhydryl group(s) and disulfide bridges
6. Carbohydrate structure
 Physicochemical properties
1. Molecular weight or size
2. Isoform pattern
3. Extinction coefficient3. Extinction coefficient
4. Electrophoretic pattern
5. Liquid Chromatographic pattern
6. Spectroscopic profiles
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15. 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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16. 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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17. 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 = +162
G2F Mass shift = +324
+ 1 x G1F
mAb +2 x G0F
mAb +1 x G1F
+ 1 x G2F
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18. INTACT MASS COMPARISON OF THREE
BIOSIMILAR MABS
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19. PEPTIDE MAPPING WORKFLOWPEPTIDE MAPPING WORKFLOW
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20. ANTIBODY ANALYSIS – GENERAL
WORKFLOW
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21. SULFHYDRYL GROUP(S) AND DISULFIDE
BRIDGES
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22. CHARACTERIZATION OF S S BRIDGESCHARACTERIZATION OF S-S BRIDGES
Disulphide bridged
2567.0100
Voyager Spec #1 MC[BP = 3017.6, 2567]
Disulphide bridged
protein
Enzymic/Chemical
di ti
S S SH
Mixture of
peptides
40
50
60
70
80
90
%Intensity
KTCIVPEVSSVFIFPPKPK
252 269
S S
SH
E
E
digestion
2971.0 2989.6 3008.2 3026.8 3045.4 3064.0
Mass (m/z)
00
10
20
30
40
C SS
KVTCVVVDISK
280 289
SH
E
E
Identification by MS
Followed by reduction
A d f th MS
Reduction
And further MS
1122.8
80
90
100
Voyager Spec #1=>SM5[BP = 1662.4, 7089]
754.3
80
90
100
Voyager Spec #1=>SM5[BP = 1662.4, 7089]
1062.6
1988.1
VTCVVVDISK
280 289
TCIVPEVSSVFIFPPKPK
252 269
30
40
50
60
70
%Intensity
30
40
50
60
70
%Intensity
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1154.0 1169.4 1184.8 1200.2 1215.6 1231.0
Mass (m/z)
20
30
1955 1970 1985 2000 2015 2030
Mass (m/z)
10
20

23. GLYCOSYLATION CHARACTERIZATIONGLYCOSYLATION CHARACTERIZATION
Structural characterisation and confirmation of carbohydrate.y
For 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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24. ANALYSIS OF GLYCOSYLATIONANALYSIS OF GLYCOSYLATION
N-Glycans Intact Mass by MALDI or
COOH2HN
S---S
S---S
N-Glycans
O-Glycans
Intact Mass by MALDI or
ES MS
Monosaccharide
Composition Analysis (LC &Reduction Carboxymethylation
MS)
COOH2HN
S-CM S-CMS-CMS-CM
Specific Protease DigestSpecific Protease Digest
PNG FPNGase F
Sep-pak Monosaccharide
Composition
Reductive
elimination
0% 20% 40%
Permethylation MALDI,
Nanospray MS/MS & Linkage analysis
Composition
Glycan Population Screening
Glycan Antennary Profile
Glycosylation Site
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Nanospray-MS/MS & Linkage analysis
LC & MS methods
Linkage Analysis

25. GLYCOMIC / GLYCOPROTEOMIC
WORKFLOW
Anomeric specific
Intermonosaccharide Glycan
C iti
MALDI-MS
Linkages Glycan Profile
HPAEC-PAD
Heterogeneity
& Extent of
Composition
Glycan
MALDI-MS
Native
Glycans
Intact mass vs.
Deglycosylated
ES MS / MALDI MS
Glycosylation
y
Sequence
MALDI-MS/MS
A t
SampleGlycoprotein
ES-MS / MALDI-MS
Quantitative
Monosaccharide
Composition
Derivatised
Glycans
Antennary
Profile
ESI-MS
Glycopeptides
Composition
GC-MS
Quantitative
PMAA GC MS
Inter-
monosaccharide
Linkages
Peptide mapping
Qualitative Site-specific
Glycosylation
HPAEC-PAD
Quantitative
Sialic Acid
Content
PMAA GC-MS
Quantitative
Glycan profile
24© SGS SA 2014 ALL RIGHTS RESERVED
LC-ES-MS
2AB-LC-MS
y p

26. OLIGOSACCHARIDE PROFILING
LC- AND MS-BASED METHODS
2-AB labelling and HPLC-FLD for profiling Oligosaccharide population coupled with ESI-MS
TIC chromatogram
Annotations based on MS dataAnnotations based on MS data
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Example of IgG N-glycans
N-acetylglucosamine
Galactose
Mannose
Fucose
N-acetylneuraminic acid
N-glycolylneuraminic acid

27. CHARGE COMPARABILITYCHARGE COMPARABILITY
Imaging cIEF ofImaging cIEF of
Monoclonal Antibodies
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28. BIOPHYSICAL TECHNIQUES FOR HIGHER
ORDER STRUCTURE CONFORMATION ANDORDER STRUCTURE, CONFORMATION AND
AGGREGATION
Technique Reports on Advantages DisadvantagesTechnique Reports on Advantages Disadvantages
Circular Dichroism
Secondary/ Tertiary
Structure
Quantitative
Sensitive to helix content
Formulation buffers can interfere
Quantitative
FTIR Secondary Structure Sensitive to sheet content
Less prone to buffer interfence
Intrinsic
Fluorescence
Local Tertiary Structure
Sensitive
Potential for moderate HTP
Qualitative
Extrinsic Fluorescence Surface hydrophobicity
Sensitive
Ensemble tertiary structure-no local
Potential for moderate HTP
Qualitative
UV-VIS (2ndderivative) Local Tertiary Structure
Simultaneous to concentration
determination QualitativeUV VIS (2 derivative) Local Tertiary Structure determination
Potential 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 present
High shear forces

29. 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/Glycomic
strategies 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
28© SGS SA 2014 ALL RIGHTS RESERVED
 Increasing importance on HOS to link with biological activity.

30. THANK YOU FOR YOUR ATTENTIONTHANK YOU FOR YOUR ATTENTION
Life Science Services Fiona Greer
Global Director, BioPharma Services Development
SGS M Scan Ltd Phone: +44 (0) 118 989 6940
+ 41 22 739 9548SGS M-Scan Ltd Phone: +44 (0) 118 989 6940
2-3 Millars Business Centre, Fax: +44 (0) 118 989 6941
Fishponds Close,
Wokingham E-mail : fiona.greer@sgs.com
Berkshire, 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