This document discusses regulatory guidelines for biosimilar recombinant proteins and their clinical use. It provides an overview of the complexities of biosimilars due to their molecular structure and manufacturing processes. The "totality of evidence" and "fingerprint" approaches are used to demonstrate biosimilarity through comparative exercises of quality attributes, non-clinical studies, and clinical trials. The level of evidence required varies depending on the complexity of the biological product and indication. Extrapolation of clinical data to other indications may be possible depending on these factors.

1. Biosimilar Recombinant Proteins and
the Current Regulatory Guidelines for
Clinical Use
Massimo Iacobelli, MD, PhD,
Consultant on Pharmaceutical Strategic Planning and
Development, Loyola University Chicago, Chicago, USA
1
Recombinant Coagulation Factors and their
Biosimilar Counterparts. A Case of Factor VIIa in
the Management of Hemophilia with Inhibitors.
Symposium in cooperation with the International Union of Angiology
24th International Congress on Thrombosis, Istanbul 6 May 2016

2. Global Biosimilars Clinical Development Activity
2

3. Biosimilars
Biological medicinal products are usually more
difficult to characterise than chemically derived
medicinal products.
• There is a spectrum of molecular complexity among the various
products (recombinant proteins, mAb, blood or plasma-
derived, immunologicals, etc.).
• Parameters such as the three-dimensional structure, or
post-translational modifications such as the glycosylation
profile can be significantly altered by changes in
manufacturing process.
3

4. Inherent high level of complexity with
Biosimilars
High Technical Hurdles
• “Stepwise”, “Fingerprint”, “Totality of Evidence” Approaches
• Comparability Exercise
• Clone, Cell line development, Master Cell Bank
• Process Development
• Manufacturing, scale-up, tech transfers, process control, Quality
• IP landscape
• PK/PD studies vs clinical trials
• Clinical trial design in the absence of a strong therapeutic signal
• Bioequivalence vs non-inferiority designs
• Clinical trial operations and patient recruitment given biosimilars
4

5. 5
FDA follows a step-wise approach to
demonstrate similarity
• FDA approved the first biosimilar in March 2015: Zarxio (filgrastim).
• The first mAb, Inflectra (infliximab) has been approved in April 2016

6. Biosimilars in Europe
The environment and experience to date in Europe remains
positive for Biosimilars.
 Up to now EMA approved 20 Biosimilars, including the
Inflectra (Infliximab), the first Biosimilar monoclonal
antibody approved in 2013
 One time Approval by EMA for 28 countries
 Use guidance (Biosimilar guidelines for industry)
 Scientific advice from EMA
 CHMP Working Party on Biosimilars
 Product development and dossiers remain complex –
there is no magic shortcut!
6

7. Manufacturing
Process
Required Data for biosimilar approval
Innovative Product Biosimilar
Characterization
of Quality
Attributes
Non clinical
studies
Clinical
studies
Manufacturing
Process
Characterization
of Quality
Attributes
Non clinical
studies
Clinical
studies
Establish its own
manufacturing
process
Characterization,
Comparability
exercise
Comparability/ Similarity
Except for:
• safety pharmacology
• reproductive pharm.,
• carcinogenicity, ecc
Comparability/ Similarity
• PK/PD,
• Efficacy, Safety
7

8. Comparability Exercise
Within the European Union regulatory framework, the
primary objective in evaluating a Biosimilar application is to
determine the similarity (or not) of a given biological
medicinal product to a reference medicinal product.
The “comparability exercise” should be a robust head-to-
head comparison between the similar biological medicinal
product and the reference medicinal product (RMP)
performed at the levels of quality, safety and efficacy.
Due to the diversity of biological medicinal products, the
assessment of biosimilar products should be done on a
case-by-case basis.
8

9. 9
Comparability exercise
 Assessment of composition, physical properties,
primary and higher order structures, purity, product-
related isoforms and impurities, and biological activity
 Use of a wide range of sensitive analytical methods
 Target amino acid sequence should be confirmed and is
expected to be the same as for the reference product
 Performed with finished product (DP)
 Any difference in the quality attributes require a satisfactory
justification of the potential implications with regard to
safety and efficacy.

10. Post translational modifications:.:
• NP-HPLC-(MS) N-glycans
• AEX N-glycans
• MALDI-TOF N-glycans
• HPAEC-PAD N-glycans
• MALDI-TOF O-glycans
• HPAEC-PAD sialic acids
• RP-HPLC sialic acids
Primary structure :
• LC-MS intact mass
• LC-MS subunits
• Peptide mapping
Higher order structure:
• NMR
• CD spectroscopy
• FT-IRImpurities:
• CEX, cIEF acidic/basic
variants
• LC glycation
• Peptide mapping
deamidation,
• oxidation, mutation, glycation
• SEC/FFF/AUC aggregation
Combination of attributes:
• MVDA, mathematical algorithms
Biological activity:
• Target binding
• Activity
• Fc receptor binging
• ADCC
• CDC
• Apoptosis
• In vitro immunogenicity
10EMA Workshop on Biosimilars, London 31 October 2013
The Fingerprint approach
Performed with finished product (DP),
comparing biosimilar with originator (RMP)

11. 11
Target ranges for assessing biosimilarity
Target ranges:
•defined by lot-to-lot
variation of quality
attributes & assay
accuracy
•to be based on “as
many as possible”
originator lots
Characterization of the “Originator” (RMP) to define a
"corridor“, representing its inherent variability
MedianStd Dev Std Dev
OutlierOutlier
**
Definition of project goals before the project start
Quality
difference

12. Level of Compliance: Significant Quality
Difference
A quality difference exists where a quality attribute is
observed to be outside the target acceptance criteria.
In the event that quality differences cannot reasonably be
avoided, sponsors should be recommended to promptly
seek scientific advice with EMA to confirm:
That for less significant differences,
the proposed analytical, nonclinical
and clinical program is sufficient to
demonstrate safety and efficacy in
all indications sought.
That the difference is not
considered a significant
quality difference that may
effectively preclude
development as a biosimilar.
12

13. EMA Guideline - Non-clinical aspects
• The 3R principles should always be considered.
Factors to be
considered:
• Presence of relevant quality attributes not
detected in the RP → e.g. new post-translational
modification structures
• Significant quantitative differences in quality
attributes
• Relevant differences in formulation
PK and/or PD
studies:
When the model allows, test and RP should be
quantitatively compared (e.g. by concentration-
response assessment
Safety
studies:
A flexible approach should be considered, in particular
if non-human primates are the only relevant species
→ e.g. in-life evaluation of safety parameters, just one
dose level, just one gender, no recovery animals
Determination of the need for in vivo animal studies

14. Clinical Development of Biosimilars
Considerable Points in the Clinical Development of
Biosimilars
• Overall clinical program: PK/PD + Phase III vs PK/PD only
• Phase I trial (PK/PD or PK study)
• Target subject: patients or healthy volunteers
• PD markers
• Relationship between PK parameters and
immunogenicity
• Phase III trial (Efficacy and Safety)
• Target indication
• Primary efficacy endpoint/s
• Bioequivalence vs non-inferiority
• Safety, immunogenicity
• Extrapolation of data to other indications
14

15. 15
In specific circumstances, e.g. for structurally more
simple biological medicinal products, a comparative
clinical efficacy study may not be necessary if similarity
of physicochemical characteristics and biological
activity/potency of the biosimilar and the reference
product can be convincingly shown and similar efficacy
and safety can clearly be deduced from these data and
comparative PK data.
Such an approach may have to be supported by additional
data, for example in vitro and/or clinical PD data from a
comprehensive comparative PD fingerprint approach.
EMA, Tailored Development Program
EMA Workshop on Biosimilars, London 31 October 2013

16. 16
• Molecular complexity
• How well mechanism of action and targets of innovator
have been characterized
• Molecular weight
• Nature of the indication and patient population
• Safety experience of the innovator product
• Complexity of endpoint of pivotal trials for innovator
• Biological class
• Immunogenicity of the Reference Product
• Existence of reliable biomarkers
Factors influencing clinical trials

17. BiosimiarsApprovedinEurope
17
Name
Active
substance
Company Clinical trials for Approval
Number
Patients
Authorizatio
n/ refusal
Abasaglar insulin glargine Eli Lilly 5 phase I and 2 phase III studies. 1475 09/09/2014
Abseamed epoetin alfa
Medice
Arzneimittel
5 pharmacology PK/PD studies in healthy volunteers,
1 confirmative phase III trial and 1 exploratory study
728 28/08/2007
Accofil f
filgrastim Eli Lilly
4 comparative PK/PD Phase I studies and 1 Phase III
non comparative, multicentre, repeat dose safety study
355 18/09/2014
Bemfola follitropin alfa Finox Biotec
1 Phase I study in healthy subjects and 1 Phase III study
in infertile ovulatory women undergoing ART.
420 27/03/2014
Binocrit epoetin alfa Sandoz Same data of Abseamed 728 28/08/2007
Biograstim filgrastim AbZ-Pharma 2 phase I studies, 3 phase III studies 869 15/09/2008
Epoetin Hexal epoetin alfa Hexal Same data of Abseamed: 728 28/08/2007
Filgrastim Hexal filgrastim Hexal 4 PK/PD studies Phase I studies in healthy volunteers 316 06/02/2009
Grastofil filgrastim Apotex Same data of Accofil. 355 18/10/2013
Inflectra infliximab Hospira 2 Phase I PK and 1 Phase III effcacy in RA patients 875 10/09/2013
Nivestim filgrastim Hospira
2 phase I PK/PD, single-centre, healthy volunteer and 1
phase III, multicentre, double-blind efficay study
342 08/06/2010
Omnitrope somatropin Sandoz 3 Phase I PK studies and 2 Phase III studies 201 12/04/2006
Ovaleap follitropin alfa Teva
2 Phase I studies in healthy subjects and a Phase III
study in infertile ovulatory women undergoing ART
375 27/09/2013
Ratiograstim filgrastim Ratiopharm Same data Biograstim 869 15/09/2008
Remsima infliximab Celltrion Same data of Inflectra 875 10/09/2013
Retacrit epoetin zeta Hospira
2 PK Phase I studies in healthy volunteers, 2 Phase III
clinical studies and 1 uncontrolled safety trial.
1210 18/12/2007
Silapo epoetin zeta Stada AG Same data of Retacrit 1210 18/12/2007
Tevagrastim filgrastim Teva Same data of Biograstim and Ratiograstim. 869 15/09/2008
Zarzio filgrastim Sandoz Same data of Filgrastim Hexal 316 06/02/2009

18. 18
Immunogenicity
• State of the art immunogenicity testing a key to show
Clinical safety
How much clinical immunogenicity data is required to
support registration?:
• At least one directly comparative study to
demonstrate immunogenicity of candidate product is
not increased vs reference.
• Ideally, with highest sensitivity to detect adverse outcomes
• Exclude subjects previously treated with reference product, or
pre-specify a subgroup analysis for previously treated subjects
• Monitor ADA formation

19. Conclusions
• Biologic medicines play a significant role in patient care
across a growing number of disease areas.
• The prospect of Biosimilars that are safe and effective
opens up opportunities for health systems to expand
access to biologics for more patients, free up resources
for investment in new areas, and bring relief to pressured
healthcare budgets.
• By 2020, the cumulative potential savings to health systems in
the five major European Union (EU) markets and the U.S., as a
result of the use of biosimilars, could exceed EUR50 billion in
aggregate over the next five years and reach as much as
EUR100 billion.
19