The development pathway of a biosimilar is unlike that of a novel biotherapeutic. While there is an increased requirement for analytics throughout a biosimilars development project, and a Phase II clinical trial is generally omitted, careful consideration must be given to the planning of the other phases of development. Many regulatory authorities reference a “step-by-step” approach to establishing biosimilarity. This presentation will provide a look at the multi-stage development for a biosimilar, including a review of the regulatory landscape, structural characterization techniques for biosimilarity assessment, and early phase clinical research challenges

1. BIOSIMILAR DEVELOPMENT:
REGULATORY, ANALYTICAL, AND
CLINICAL CONSIDERATIONS
SGS LIFE SCIENCE SERVICES WEBINAR
CLINICAL CONSIDERATIONS
April 22, 2015

2. MEETING REGULATORY ANALYTICAL
CHARACTERIZATION EXPECTATIONS
BIOSIMILAR DEVELOPMENT:
REGULATORY, ANALYTICAL, AND CLINICAL CONSIDERATIONS
Dr Fiona M Greer
Global Director, BioPharma Services Development
SGS Life Science Services

3. AGENDA: MEETING REGULATORY ANALYTICAL
CHARACTERIZATION EXPECTATIONS
What are the challenges in characterizing complex
protein/glycoprotein products?
When is analytical characterization required?
Which techniques, old & new, are suitable
• Establishing the Quality Target Product Profile (QTPP)
3© SGS SA 2015 ALL RIGHTS RESERVED
• Package of analytical tools/ battery of methods
• Strategies for primary and higher order structure

4. WHY ARE BIOPRODUCTS A CHALLENGE ?
During/ after translation of the oligonucleotide
code into an AA sequence, processing events
occur to confer biological activity. These Co-
and Post-Translational events change the
primary structure but are NOT predictable from
the gene sequence. So it is essential to study
the expressed protein products not the genes.
Acetylation
Acylation
Amidation
(deamidation)
Carbamylation
Carboxylation
Formylation
Glycation
Glycosylation
Methylation
Methionine Oxidation
Norleucine
Phosphorylation
Sulphation
Carbohydrate is a source of product
4© SGS SA 2015 ALL RIGHTS RESERVED
Carbohydrate is a source of product
heterogeneity. Glycoproteins are mixtures of
glycoforms i.e. the same polypeptide but
different sugars.

5. EXAMPLE OF COMPLEXITY: ANTIBODY CASE
5© SGS SA 2015 ALL RIGHTS RESERVED

6. WHEN IS ANALYTICAL CHARACTERIZATION
REQUIRED?
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7. WHAT REGULATIONS COVER
PHYSICOCHEMICAL CHARACTERIZATION?
ICH Topic Q6B “Specifications: Test Procedures
and Acceptance Criteria for
Biotechnological/Biological Products”
Structural characterization and confirmation
1. Amino acid sequence
2. Amino acid composition
3. Terminal amino acid sequence
7© SGS SA 2015 ALL RIGHTS RESERVED
3. 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 coefficient
4. Electrophoretic pattern
5. Liquid Chromatographic pattern
6. Spectroscopic profiles

8. POTENTIAL ANALYTICAL TOOLS
Amino acid sequence and modifications: MS, peptide mapping,
chromatography
Glycosylation: Anion exchange, enzymatic digestion, peptide
mapping, CE, MS
Folding: MS S-S bridge determination, calorimetry, HDX and ion
mobility MS, NMR, circular dichroism, Fourier transform
spectroscopy, fluorescence
8© SGS SA 2015 ALL RIGHTS RESERVED
spectroscopy, fluorescence
PEGylation & isomers: chromatography, peptide mapping
Aggregation: Analytical ultracentrifugation, size-exclusion
chromatography SEC-MALS, field flow fractionation A4F, light
scattering DLS, microscopy, TEM
Proteolysis: electrophoresis, chromatography, MS
Impurities: proteomics, immunoassays, metal & solvents analysis
Subunit interactions: chromatography, ion mobility MS
Heterogeneity of size, charge, hydrophobicity: Chromatography;
gel & capillary electrophoresis, light scattering, IM-MS, CESI-MS

9. CASE STUDY: ANTIBODY CHARACTERIZATION
• Mass spectrometry of intact
protein and released L &H chains
• Amino Acid Composition Analysis
• N- and C-terminal sequencing
• Peptide “MAPPING” Analysis
(Sequence coverage: 100% LC
and 100% HC)
9© SGS SA 2015 ALL RIGHTS RESERVED
and 100% HC)
• Monosaccharide and sialic acid
analysis
• Oligosaccharide population
analysis
• SDS-PAGE analysis
• Circular Dichroism
• Analytical Ultracentrifugation

10. N-Linked biantennary core fucosylated with varying number of galactose residues
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
INTACT MASS MEASUREMENT (MONITORING
GLYCOSYLATION)
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mAb +2 x G0F
+ 1 x G1F
mAb +2 x G1F
mAb +1 x G1F
+ 1 x G2F

11. INTACT MASS COMPARISON OF THREE
BIOSIMILAR MABS
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12. PEPTIDE MAPPING WORKFLOW
12© SGS SA 2015 ALL RIGHTS RESERVED

13. ANTIBODY ANALYSIS – GENERAL WORKFLOW
13© SGS SA 2015 ALL RIGHTS RESERVED

14. SULFHYDRYL GROUP(S) AND DISULFIDE
BRIDGES
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15. S S
SH
Disulphide bridged
protein
E
E
Enzymic/Chemical
digestion
S S SH
Mixture of
peptides
2971.0 2989.6 3008.2 3026.8 3045.4 3064.0
Mass (m/z)
0
2567.0
0
10
20
30
40
50
60
70
80
90
100
%Intensity
Voyager Spec #1 MC[BP = 3017.6, 2567]
KTCIVPEVSSVFIFPPKPK
KVTCVVVDISK
252 269
280 289
CHARACTERIZATION OF S-S BRIDGES
15© SGS SA 2015 ALL RIGHTS RESERVED
SH
E
E
Identification by MS
Followed by reduction
And further MS
1154.0 1169.4 1184.8 1200.2 1215.6 1231.0
Mass (m/z)
1122.8
20
30
40
50
60
70
80
90
100
%Intensity
Voyager Spec #1=>SM5[BP = 1662.4, 7089]
1955 1970 1985 2000 2015 2030
Mass (m/z)
754.3
10
20
30
40
50
60
70
80
90
100
%Intensity
Voyager Spec #1=>SM5[BP = 1662.4, 7089]
1062.6
1988.1
VTCVVVDISK
280 289
TCIVPEVSSVFIFPPKPK
252 269
Reduction

16. ANALYSIS OF GLYCOSYLATION
COOH2HN
S---S
S---S
N-Glycans
O-Glycans
Intact Mass by MALDI or ES
MS
Monosaccharide Composition
Analysis (LC & MS)Reduction Carboxymethylation
COOH2HN
S-CM S-CMS-CMS-CM
Specific Protease Digest
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Reductive
elimination
Specific Protease Digest
PNGase F
Sep-pak
0% 20% 40%
Permethylation MALDI,
Nanospray-MS/MS & Linkage analysis
LC & MS methods
Monosaccharide Composition
Glycan Population Screening
Glycan Antennary Profile
Glycosylation Site
Linkage Analysis

17. Native
Glycans
MALDI-MS
Anomeric specific
Intermonosaccharide
Linkages Glycan Profile
HPAEC-PAD
Intact mass vs.
Deglycosylated
ES-MS / MALDI-MS
Heterogeneity
& Extent of
Glycosylation
Derivatised
Glycan
Composition
Glycan
Sequence
MALDI-MS
MALDI-MS/MS
Antennary
GLYCOMIC / GLYCOPROTEOMIC WORKFLOW
17© SGS SA 2015 ALL RIGHTS RESERVED
SampleGlycoprotein
ES-MS / MALDI-MS
Quantitative
Monosaccharide
Composition
GC-MS
HPAEC-PAD
Quantitative
Sialic Acid
Content
Derivatised
Glycans
PMAA GC-MS
Inter-
monosaccharide
Linkages
Antennary
Profile
ESI-MS
2AB-LC-MS
Quantitative
Glycan profile
Glycopeptides
Qualitative Site-specific
Glycosylation
Peptide Mapping
LC-ES-MS

18. MALDI-MS analysis of permethylated N-glycans
MAJOR STRUCTURES OF N-LINKED
OLIGOSACCHARIDES ON IGG1 MABS
Possible structure for the signal at m/z 1836 (G0F)
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Possible structure for the signal at m/z 2040 (G1F)
Possible structure for the signal at m/z 2244 (G2F)

19. TIC chromatogram
Annotations based on MS data
OLIGOSACCHARIDE PROFILING
LC- AND MS-BASED METHODS
2-AB labelling and HPLC-FLD for profiling Oligosaccharide population coupled with ESI-MS
19© SGS SA 2015 ALL RIGHTS RESERVED
Annotations based on MS data
Example of IgG N-glycans
N-acetylglucosamine
Galactose
Mannose
Fucose
N-acetylneuraminic acid
N-glycolylneuraminic acid

20. BIOPHYSICAL TECHNIQUES FOR HIGHER ORDER
STRUCTURE, CONFORMATION AND
AGGREGATION
Technique Reports on Advantages Disadvantages
Circular Dichroism
Secondary/ Tertiary
Structure
Quantitative
Sensitive to helix content
Formulation buffers can interfere
FTIR Secondary Structure
Quantitative
Sensitive to sheet content
Less prone to buffer interfence
Intrinsic
Fluorescence
Local Tertiary Structure
Sensitive
Potential for moderate HTP
Qualitative
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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
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
SEC-MALS
Oligomers/ aggregates
Direct MW determination, rapid analysis
Matrix present
High shear forces

21. RE-CAP: ANALYTICAL CHARACTERIZATION
DATA FOR BIOSIMILARS
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 attributes. These
data form the Quality Target Product Profile (QTPP).
MS techniques are applicable at all stages of development, but
essential for determination of originator sequence. Advances in
21© SGS SA 2015 ALL RIGHTS RESERVED
essential for determination of originator sequence. Advances in
instrumentation and Proteomic/Glycomic strategies 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”.
MS alone is not enough. Multiple orthogonal analytical methods are
used to define “fingerprint” comparison.
Increasing importance on HOS techniques to link with biological
activity.

22. Life Science Services Fiona Greer
Global Director, BioPharma Services Development
SGS M-Scan Ltd Phone: +44 (0) 118 989 6940
THANK YOU FOR YOUR ATTENTION
+ 41 22 739 9548
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SGS 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
+ 41 22 739 9548
+ 1 866 SGS 5003
+ 65 637 90 111
+ 33 1 53 78 18 79
+ 1 877 677 2667
+ 33 1 41 24 87 87

23. MEETING THE CHALLENGES OF EARLY
PHASE CLINICAL TRIALS WITH
BIOSIMILARS
BIOSIMILAR DEVELOPMENT:
REGULATORY, ANALYTICAL, AND CLINICAL CONSIDERATIONS
BIOSIMILARS
Annick Van Riel
Director of the Clinical Pharmacology Unit
SGS Life Science Services

24. OVERVIEW
Opportunities
Regulatory Challenges
Comparative Clinical Trials
• a Stepwise Approach
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• a Stepwise Approach
Clinical Considerations in phase I
• Operational Challenges
• Operational Solutions

25. OPPORTUNITIES
Market for Biosimilars will reach $9,2bn in 2018
Key drivers
• Patent expiry of biologic drugs
– By 2020, some $55bn biologic patents due to expire
• Increasing market demands
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• Increasing market demands
– Aging population
– Health awareness
– Affordability and insurance coverage
• Increasing healthcare cost
• Legislations (US, EU, Asia)
• Competitive landscape
Affordable, safe, efficacious biological drugs

26. REGULATORY CHALLENGES
- REFERENCE PRODUCT
SELECTION of REFERENCE PRODUCT (RBP)
• Market (Europe / US / Both)
• Indication
– Extrapolation across other indications of the RBP will need
scientific justification
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COMPARABILITY in SAFETY AND EFFICACY
• Deviations from RBP (strength, pharmaceutical form,
formulation,…) require justification
– Route of administration
– Improved efficacy
– Improved safety

27. REGULATORY CHALLENGES
- DOSSIER
NON-CLINICAL DEVELOPMENT
• ‘Cutting corners’ on EU / FDA guidance on non-clinical
development
• Risk, but can be acceptable if product is considered safe
Chemistry, Manufacturing & Controls (CMC)
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Chemistry, Manufacturing & Controls (CMC)
• CMC document should clearly demonstrate similarity
• Comparability exercise well presented
PERCEIVED RISKS of biosimilars
• Supportive EC that understands what a biosimilar is
• Scientific Advice meeting with Regulatory Authority
(FDA/EMA/…)

28. COMPARATIVE CLINICAL TRIALS
- A STEPWISE APPROACH
Clinical evaluation in
intended population
Non-clinical testing
Analytical comparison
to reference product
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to reference product
• Structure
• Function
Each step supported by
preceding one.
Published in: Mark A Socinski; Giuseppe Curigliano; Ira Jacobs; Barry Gumbiner; Judith MacDonald;
Dolca Thomas; mAbs 2015, 7, 286-293.

29. COMPARATIVE CLINICAL TRIALS
- A STEPWISE APPROACH
CLINICAL DEVELOPMENT
To show comparable safety and efficacy
between RBP / SBP
• Detect and explore relevant differences
•
29© SGS SA 2015 ALL RIGHTS RESERVED
• Specific indication
• 3-arm trial design
• STEP 1A – Pharmacokinetics
– Homogenous population, eg healthy volunteers
– Supportive PK data from patients
– Selected, single dose
» Except where dose / time dependent PK
– Similarity acceptance range should be pre-defined (80-125%)

30. COMPARATIVE CLINICAL TRIALS
- A STEPWISE APPROACH
CLINICAL DEVELOPMENT
• STEP 1B – Pharmacodynamics
– Clear dose response relationship
– Accepted PD markers
– Population in which the possible differences could be best observed
30© SGS SA 2015 ALL RIGHTS RESERVED
– Population in which the possible differences could be best observed
• Comparative PK / PD trial may be sufficient
– PK of RBP well characterized
– Sufficient knowledge on PD properties / action mechanism
– One PD marker is linked to efficacy
– Dose/exposure and response/efficacy of RBP is well established
To be discussed with EMA / FDA during Scientific Advise

31. COMPARATIVE CLINICAL TRIALS
- A STEPWISE APPROACH
CLINICAL DEVELOPMENT
• STEP 2A – Clinical Efficacy
– Designed to prove similar clinical efficacy between RBP/SBP
» Similar treatment effect
» Similar dosage
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– Similarity acceptance range should be pre-defined
– Choose most sensitive population
– Extrapolation of indication possible based on overall evidence of
biosimilarity
• STEP 2B – Clinical Safety
– Type, severity, frequency of ADRs between RBP/SBP
– Assessment of immunogenicity
– Pharmacovigilance and risk management plan

32. CLINICAL TRIAL CONSIDERATIONS IN PHASE I
- OPERATIONAL CHALLENGES
Comparability – NOT Characterization
PK and PD endpoints (primary / secondary)
Large inter and intra subject variability
Bridging with US and EU reference
• 3-arm studies
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• 3-arm studies
Large sample size
Study design
• Cross over or parallel
• Half life
• Safety
• Dose (single / multiple …)
• Driven by PD, not PK

33. CLINICAL TRIAL CONSIDERATIONS IN PHASE I
- OPERATIONAL CHALLENGES
Biosimilar experience
Bioanalytical
knowledge
Quality – control
variability
Regulatory approval
Number of cohorts, bed
size
Population -
Recruitment
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variability
Pharmacy IMP
preparation
Clinical Conduct
Sample processing and
shipment
Recruitment
Screening
Staffing
Timelines

34. Randomized, double-blind, 3-way parallel study
to compare PK between X and adalimumab (EU
and US sourced).
• PK, safety, tolerability
• Subcutaneous injection
1
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• Subcutaneous injection
• Single center, 252 healthy male / female volunteers
CHALLENGE
• Regulatory
– Submission rejected in The Netherlands
• Recruitment
• Screening
• Timelines

35. Randomised, double-blind, three-arm, parallel
group, single dose study to compare the PK,
safety, tolerability and immunogenicity of X and
reference product (EU and US sourced) in
healthy male subjects.
• PK, safety, tolerability, immunogenicity
2
35© SGS SA 2015 ALL RIGHTS RESERVED
• PK, safety, tolerability, immunogenicity
• IV infusion
• Single center, 147 healthy male volunteers, 120 days
CHALLENGE
• Recruitment
• Screening
• Timelines

36. CLINICAL TRIAL CONSIDERATIONS IN PHASE I
- OPERATIONAL SOLUTIONS
REGULATORY SOLUTIONS
• CMC file extensively reworked, including head to head
comparison of analytical data
• Recent experience in Belgium (15 biosimilar trials)
– 100% approval rate for biosimilar studies
36© SGS SA 2015 ALL RIGHTS RESERVED
– CA comments
» Clarification on manufacturing sites
» Recommendation on in vitro assay in non-clinical program
– IRB/EC comments
» None received

37. CLINICAL TRIAL CONSIDERATIONS IN PHASE I
- OPERATIONAL SOLUTIONS
POPULATION SOLUTIONS
• Sponsor preference = biosimilar naïve subjects
• Belgium CA allow phase I biosimilar studies to be
performed in non-biologic naïve subjects.
– Appropriate wash-out times to be observed
37© SGS SA 2015 ALL RIGHTS RESERVED
• Continuous building of CPU healthy volunteer
database to expand biologic-naïve population, due to
sponsor preferences.

38. CLINICAL TRIAL CONSIDERATIONS IN PHASE I
- OPERATIONAL SOLUTIONS
RECRUITMENT and SCREENING SOLUTIONS
• Contracted Call Centre
• Advertisement
• Sources
• Sufficient volunteer fee, linked to duration
• Start immediately after EC approval
38© SGS SA 2015 ALL RIGHTS RESERVED
• Start immediately after EC approval
• Line up
– Subject availability for multiple groups
– Reserve subjects (4 per group)
• Plan
– Optional screening dates scheduled up front
– Optional groups
– Protocol to allow for re-screening of subjects
• Communicate
– Close liaison with investigator and sponsor

39. CLINICAL TRIAL CONSIDERATIONS IN PHASE I
- OPERATIONAL SOLUTIONS
TIMELINE SOLUTIONS
• Short approval timelines in Belgium
– 15 day HA/IRB(EC) review timelines
– Generic screening before CA approval High number of subjects
on short # weeks
• Line-up for screening
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• Line-up for screening
• Continuous inclusion
• Fast data processing
– Continuous data processing (on-site and data management)
– Fast database lock due to flexible teams

40. CONCLUSION
Biosimilar Clinical Development in phase I has
challenges
• High Clinical cost, resource and time
– Plan complete programme, not 1 study
• No “one-size fits all” approach
40© SGS SA 2015 ALL RIGHTS RESERVED
• No “one-size fits all” approach
• Manufacturing complex and challenging
• Regulatory requirements
– Consult regulatory bodies at planning stage
• Phase I site experience
– Subject recruitment
– timelines

41. QUESTIONS
Speakers:
Nadine M. Ritter, Ph.D.
Global Biotech Experts
Nadine.Ritter@GlobalBiotechExperts.com
Dr. Fiona M. Greer
SGS Life Science Services
fiona.greer@sgs.com
41© SGS SA 2015 ALL RIGHTS RESERVED
fiona.greer@sgs.com
Annick Van Riel
SGS Life Science Services
annick.vanriel@sgs.com
For additional information visit:
WWW.SGS.COM/BIOSIMILARS
Thank you for attending this event.