FOCUS  ON...
THE REGS
Biosimilar Therapeutic
Monoclonal Antibodies
Gaps in Science Limit Development of an Industry Standard
for Their Regulatory Approval, Part 1
Simran J. Kaur, Darryl Sampey, Lester W. Schultheis, Leonard P. Freedman, William E. Bentley
B
iosimilars are biologically
derived pharmaceuticals
intended to have clinical
similarity to a legally marketed
innovator product when that product’s
patent or market exclusivity has
expired. By contrast with generic
small-molecule drugs, clinical
performance of a biologic
pharmaceutical is a function of its
structural complexity and higher-order
structure (HOS). Biomanufacturing
controls of such complex products
cannot fully ensure chemical similarity
between an innovator product and
putative biosimilar because minor
H
differences in chemical modifications
and HOS can significantly alter a
IT
product’s safety and efficacy.
Therefore, to substantiate claims of
W
clinical functionality, a demonstration
of bioequivalence is inadequate for
biosimilar pharmaceuticals. This is
T
different from regulatory approval for
generic drugs, in which bioequivalence
IN
demonstration is adequate. The overall
challenge in approving biosimilar
pharmaceuticals is to enable scientific
R
inference of similarity in safety and
efficacy for a new biologically derived
P
product compared with an innovator
without repeating burdensome clinical
E
studies.
R
In Part 1 of this two-part article, we
focus on the scientific gaps that
complicate determination of
biosimilarity for one subset of
biologically manufactured products:
12 BioProcess International 14(9) O ctober 2016
S
Fab
IS
Hinge
M variable domain (VH). The Fab is the antigen-
S–S bonds
CH 2
C H2
Fc
R
E
P
therapeutic monoclonal antibodies
(TMAbs). Notably, many biosimilar
products have been approved around
the world, including in the United
States: e.g., Sandoz’s Zarxio biosimilar
to Amgen’s Neupogen (filgrastim) and
Celltrion/Pfizer’s Inflectra biosimilar
to Janssen/J&J’s Remicade (infliximab).
First we briefly describe the regulatory
landscape for the development of
biosimilar TMAbs. Then we identify
certain specific structural components
of TMAbs — drug substances in Part
1, drug products in Part 2 — that
warrant particular attention because
alterations to them are likely to affect
therapeutic safety and effectiveness.
Finally, in Part 2 we consider whether
studies of TMAb reference material
can further the development of
analytical industry standards to ensure
comparability of putative biosimilar
TMAbs with innovator TMAbs. And
we suggest that the time is right to tie
LY
N
O
N
IO
Figure 1:  Immunoglobulin G (IgG) structure
consists of four polypeptide chains, including two
identical light chains (~25 kDa) and two identical
heavy chains (~50 kDa). Each light chain consists of
one constant domain (CL) and one variable domain
(VL), and each heavy chain consists of three
constant domains (CH1, CH2, and CH3) and one
binding region containing hypervariable or
complementarity-determining regions (CDRs),
whereas the Fc region is highly conserved across
molecular species. The hinge region contains two
disulfide bonds and glycosylation (shown as red
dots) at CH2 domain of Fc fragment.
analytical industry standards and
manufacturing controls to specific
reductions in preclinical and clinical
studies for regulatory approval of
certain biosimilar TMAbs.
US Regulation of
Biosimilar TMAbs
Biosimilars or “follow-on biologics” are
potentially cost-effective alternatives to
innovator biopharmaceuticals because
they can significantly reduce the time it
takes for product and process
development with a lowered regulatory
burden (1, 2). The FDA approved the
first TMAb in 1986 (3), and about 30
TMAb products now account for
annual sales in excess of US$60 billion
(Table 1) (4). Pursuing the biosimilar
path rather than using the original
innovator product could save 15–30%
or more in revenues (5–7).
Many innovator biologics will reach
patent or market exclusivity expiry
 Table 1:  Top-selling therapeutic monoclonal antibody (MAb) products in 2013
Antibody/Brand Name Target
Adalimumab/Humira TNF-α
Etanercept/Enbrel TNF-α
(fusion protein)
Infliximab/Remicade TNF-α
Rituximab/Rituxan, CD-20
MAbThera
Bevacizumab/Avastin VEGF
Trastuzumab/Herceptin HER2
Ranibizumab/Lucentis VEGF
TNF = tumor necrosis factor; RA = rheumatoid arthiritis; VEGF = vascular endothelial growth factor;
HER2= human epidermal growth factor receptor 2
within the next few years (5, 6). In
anticipation, US policy has been
established to promote biosimilar
approvals. Biosimilars are defined by
the US law under Section 351(k) of
the Public Health and Service Act
(PHSA) as biologic products that are
“highly similar to the reference
product notwithstanding minor
differences in clinically inactive
components,” and without “clinically
meaningful differences between the
biological product and the reference
product in terms of the safety, purity
and potency of the product” (7).
Regulatory bodies had to consider
scientific evaluation of comparability
in pharmaceuticals long before
biosimilars were considered as
potential low-cost alternatives to
innovator products (11, 12). The advent
of biosimilars increases evaluation
complexity. During innovator-product
reviews such as for TMAbs, the issue
of clinical comparability naturally
arises because subtle manufacturing
variations may yield glycosylation
alterations or other posttranslational
modifications to the TMAb product
(13). Subtle molecular variations
(microheterogeneities) can affect
product potency and/or toxicity.
Fortunately, industry sponsors and
regulatory reviewers already have
experience in making scientific
determinations from analytical and
biological activity data. The
International Conference on
Harmonization of Technical
Requirements for Registration of
Pharmaceuticals for Human Use
(ICH) established guidelines in 2004
that describe a comparability exercise
14 BioProcess International 14(9)
2013 Sales
Company (US$ billions) Indication
Abbott Laboratories 11.00 Autoimmune
Amgen and Pfizer 8.76 Autoimmune (RA,
psoriasis, Crohn’s)
Janssen Biotech, Inc 8.37 Autoimmune
Biogen Idec and 7.91 Non-Hodgkin
Genentech lymphoma
Genentech/Roche 6.97 Colorectal cancer
Genentech/Roche 6.91 Breast cancer
Genentech and 4.27 Macular
Novartis degeneration
to ascertain whether a biologically
manufactured product retained its
quality despite changes in
manufacturing (8, 9). The 1999 ICH
Q6B guideline describes how to use
analytical procedures to establish
acceptance criteria for proteins made
by cell culture expression systems (16).
So despite manufacturing changes, an
innovator TMAb can retain its
clinical safety and efficacy previously
demonstrated in clinical studies (10).
Guidelines established by ICH Q6B
outline basic underlying principles used
to set specifications, including
characterization, analysis, and
manufacturing controls (11, 16). Further,
prespecified quantitative parameters —
e.g., primary structure, glycosylation,
disulfide structure, charge variants,
size variants, biophysical
characterization (secondary structure,
tertiary structure, and thermal stability)
,and biological characterization —
must be identified to determine
acceptability of raw materials,
excipients, and final products. Q6B
also indicates the usefulness of
pharmacopoeial tests for evaluating
biologically manufactured
pharmaceuticals in such areas as
sterility, endotoxin levels, microbial
limits, particulates, dose uniformity,
and container volume. Release and
shelf-life limits are established for
potency and degradation products from
both drug substance and finished drug
product.
In 2006, the European Medicines
Agency (EMA) published the first
regulatory guidance for biosimilar
approval (12, 13). Since then, many
biosimilars have been approved in the
O ctober 2016
European Union (14), one of which is
the TMAb Inflectra (infliximab) that
was also recently approved in the
United States (15). The EMA has now
published TMAb product-class–
specific data for production, quality
control, and nonclinical and clinical
studies (16, 17). Those guidelines
require demonstration of similarity —
in terms of quality, safety, and efficacy
— with a reference product authorized
in the European Union. Although
those products are not expected to be
identical, even minor detected
differences should be justified
scientifically with respect to the safety
and efficacy.
The World Health Organization
(WHO) published analogous guidance
in 2010 for “similar biotherapeutic
products” (SBPs) through its Expert
Committee on Biological
Standardization (ECBS) (18). We
suggest that experience and scientific
evidence gained from biosimilar
TMAbs already launched in Europe
can provide additional insight to
facilitate continuing biosimilar TMAb
approval in the United States (19).
The 2009 US Biologics Price
Competition and Innovation Act
(BPCI) and the associated 2010
Patient Protection and Affordable
Care Act (2010) incorporated a new
regulatory pathway called 351(k) for
biosimilar products (7). The US Food
and Drug Administration (FDA)
responded to those new laws by
issuing draft guidance documents for
industry sponsors and FDA reviewers
(20–26) (Table 2). According to FDA
guidelines for demonstrating
biosimilarity, a biologics license
application (BLA) will be evaluated
and reviewed under provision 351(k)
of the act for potential reduction of
nonclinical and clinical studies (27).
The agency decided on case-by-case
evaluation of clinical data based on
“totality of evidence” to approve
biosimilars. It appears that the FDA
does not accept that a standardized
analytical comparison between an
innovator and putative biosimilar
could be applied across the entire class
of TMAbs. Indeed, such a standard
might be anticipated to complicate
clinical studies initiated by sponsors.
Table 2:  US Food and Drug Administration (FDA) drafts and guidance documents on demonstrating biosimilarity

US FDA Guidance Effective

for Industry on Biosimilars
Formal Meetings Between the
FDA and Biosimilar
Biological Product Sponsors
or Applicants
Nonproprietary Naming
of Biological Products
Scientific Considerations in
Demonstrating Biosimilarity to a
Reference Product
Quality Considerations in
Demonstrating Biosimilarity to a
Reference Protein Product
Biosimilars: Questions and Answers
Regarding Implementation of the
Biologics Price Competition and
Innovation Act of 2009
Reference Product Exclusivity for
Biological Products Filed Under
Section 351(a) of the PHS Act
Date Purpose of the Guidance
November Proposes guidelines for sponsors or applicants on generating and submitting necessary
2015 documentation to the FDA to arrange meetings regarding the advancement of biosimilar
biological products
August Addresses the naming of biological products (including biosimilars) with a core name and a
2015 suffix of four lowercase letters that enables users to identify whether a product is biosimilar
or interchangeable (to minimize inadvertent substitution)
April General scientific principles for conducting comparative structural and functional analysis
2015 using orthogonal analytical methods, animal studies (including an assessment of toxicity),
human pharmacokinetic/pharmacodynamic (PK/PD) studies, clinical immunogenicity
assessment, and a clinical study or studies
April Analytical factors that should be considered when demonstrating biosimilarity, ranging from
2015 the expression system and manufacturing process to the stability of the two products,
impurities, and comparative physiochemical and functional studies
April Acceptable variations between proposed and reference products with respect to formulation,
2015 delivery device, routes of administration, extrapolation for other indications,
interchangeability, exclusivity, and alternative regulatory approach
August Information that reference product sponsors should provide (e.g., differences between its
2014 product and any predecessors) to facilitate the FDA’s determination of the date of first
licensure for their products for determining the exclusivity period of the reference product; a
biosimilar application under Section 351(k) cannot be submitted until four years after the
date of the reference product’s first licensure and cannot be approved by the FDA until 12
years after that date of first licensure.

Clinical Pharmacology Data to May Design and use of clinical pharmacology studies to support a decision that a proposed
Support a Demonstration of 2014 biological product is biosimilar to its reference product based on exposure and response
Biosimilarity to a Reference Product assessment; evaluation or residual uncertainty; and analytical quality and uncertainty

In the sections that follow — here
and in Part 2 — we describe product
and process characteristics specific to
TMAbs. We present knowledge gaps
that limit the formation of industry-
wide validation or standardization.
Thus, we will show that we agree that
the “case-by-case” nature of the
current regulatory burden cannot be
reduced at this time. Establishment of
standards for cross-class product
attributes are needed to reduce the
regulatory burden in the future.
Drug Substance:
Conserving Primary Structure
Most TMAbs are of the IgG-class of
immunoglobulins. IgGs are ~150 kDa
in size, composed of four polypeptide
chains, including two identical light
chains (~25 kDa) and two heavy chains
(~50 kDa) (Figure 1). The four
constituent polypeptides in the antibody
interact to form a three-dimensional
(3D) “Y” shape. That 3D structure
confers physiological specificity. The
Fab moiety binds antigens and contains
the complementarity-determining
region, whereas the Fc moiety is highly
conserved.
Protein structure can be described
as primary (linear amino-acid
sequence), secondary (sectional folding
of alpha helixes and beta sheets),
tertiary (3D molecular shape), and
quaternary (subunit domains
assembled into single entity).
Modification to the primary
polypeptides, such as those following
translation of the amino-acid sequence
(e.g., glycosylation, deamidation, and
degradation), can affect product safety
and efficacy (28). Among the most
noted posttranslational modifications
(PTMs) for TMAbs are their varied
N-linked glycan structures, which
include galactosylation, fucosylation,
mannosylation, and sialylation.
Glycosylation is a highly variable
and heterogeneous process that
depends on such factors as clonal
variation, production cell line, media,
and culture conditions (29, 30). We
note, however, that some changes in
glycosylation stemming from
manufacturing changes (e.g., the pre-
to postchange scenario described above)
have been accepted by FDA, allowing
for heterogeneity in glycan structures to
be accommodated (31–33). In such
cases, manufacturing process details
were made available. That information
probably would not be available to a
biosimilar TMAb sponsor intending to
compete with an innovator.
Below, we offer examples in which
glycosylation of specific regions or
portions of TMAbs could be critical
for an antibody’s pharmacologic
function. Thus, differences from an
innovator’s manufacturing controls
when a competitor attempts to create a
biosimilar TMAb can be expected to
have ramifications for therapeutic
safety and efficacy.
Fc Region of TMAbs
Example 1: N-linked glycosylation is
conserved in humans at Asn-297 of
the CH 2 domain on the Fc portion of
IgG (IgG-Fc), below the hinge region.
The IgG-Fc portion binds to Fcγ
receptors (FcγRs) on the surface of
effector immune cells, resulting in
antibody-dependent, cell-mediated
cytotoxicity (ADCC), complement-
dependent cytotoxicity (CDC),
phagocytosis, oxidative burst, and
secretion of inflammatory mediators
(34). Glycosylation of the IgG-Fc has

16 BioProcess International 14(9) O ctober 2016

been shown to be critical for
mediating the binding interaction
with FcγRs to trigger ADCC. In
particular, the lower hinge region of
IgG-Fc affects binding and activation
of FcγRIII (34, 35).
Example 2: Core fucosylation (α 1,
6-fucose linked to N-acetylglucosamine)
reduces binding affinity to the FcγRIIIa
receptor and thus degrades ADCC (36–
38). Similarly, nonfucosylated antibodies
evade the inhibitory effects of serum
IgG on ADCC through its high
binding affinity to FcγRIIIa expressed
on natural killer (NK) cells (39).
Alternatively, glycosylation at Asn-162
of the FcγRIIIa receptor is critical for
binding to the Fc region (40). For
example, MAbs produced by Chinese
hamster ovary (CHO) cells typically
have complex biatennary structures with
little bisecting N-acetylglucosamine
(GlcNAc) and a high level of core
fucosylation. Overexpression of
N-acetylglucosaminetransferase III in
such cell lines increases bisecting
GlcNAc and nonfucosylated
oligosaccharides on MAbs and thus
raises ADCC (41).
Example 3: Glycosylation of the Fc
region is also indispensable for
maintaining a long catabolic half-life
in vivo (42). IgGs containing high-
mannose glycans have shown
increased serum clearance (43) and
addition of terminal sialic acid to the
Fc glycan leads to upregulation of
surface expression of the FcγRIIb on
inf lammatory cells, thereby initiating
an antiinf lammatory cascade (44, 45).
In addition, the presence of
N-glycolylneuraminic acid (NeuGC
or NGNA) in recombinant
therapeutic glycoproteins expressed in
nonhuman cell cultures may be
immunogenic and potentially relevant
to half-life, efficacy, and adverse
events (46, 47). Also, glycosylation at
the CH 2 domain of IgG1-Fc affects
C1q/C1 binding and activation
leading to inf lammatory and
immunoregulatory responses
consistent with CDC (48).
Thus, an abundance of scientific
evidence indicates that specific
glycosylation of the Fc region can be
quantitatively evaluated (49). Fc
glycoprofiles are expected to be
18 BioProcess International 14(9) O ctober 2016
strongly associated with safety and
efficacy.
Fab Region of TMAbs
Example 4: The oligosaccharide
galactose-α-1,3-galactose on the Fab
portion of the heavy chain of
cetuximab, a chimeric IgG1 MAb
against the epidermal growth factor
receptor (EGFR), is linked to
unwanted immunological response
(50). It is also notable that 15–25% of
the IgG-Fab variable domain contains
N-linked oligosaccharides occupied by
biantennary carbohydrates with
variable sialic acid content (51, 52).
Variable-domain glycosylation on Ab
function suggests that physiological
performance may be comparably
varied (52), including a potential
relationship to Ab half-life (53).
To achieve biosimilarity of
TMAbs, their primary structure
(including posttranslational
modifications) must be rigorously
conserved. Whereas ample data are
available relating the physiologic and
clinical function of TMAbs to their
posttranslational chemistry, the body
of science is not comprehensive. So
this information cannot by itself
predict clinical outcomes.
Biosimilar developers face an even
more daunting manufacturing
challenge than innovators do when
controlling primary structure of a
T-MAb. Unlike innovator
companies, developers of biosimilar
TMAbs have no access to the
original, proprietary manufacturing
process data from the originator.
Therefore, biosimilar developers
must completely reverse-engineer
their manufacturing methods and
process controls using publicly
available scientific knowledge about
the innovator TMAbs (32). In
addition, even with a comparable
product from a reverse-engineered
manufacturing process, biosimilar
TMAbs developers must attempt to
recreate a new, therapeutically
equivalent product formulation (see
part 2) — whereas innovators simply
maintain quality control over an
approved TMAb production process
and formulation.
Drug Substance
Biosimilarity Confirmation
Analysis of TMAb higher-order
structure (HOS) is just as critical as
primary structure when developers
want to establish biosimilarity. Protein
misfolding, for example, can have an
impact on drug safety by eliciting
unwanted immune responses or other
off-target physical responses. A number
of biophysical techniques are used to
characterize TMAb HOS: e.g., X-ray
crystallography, nuclear magnetic
resonance (NMR) imaging, Fourier-
transform infrared (FTIR)
spectroscopy, circular dichroism (CD),
differential scanning calorimetry
(DSC), and isothermal calorimetry.
Because no single analytical assay is
sufficient to determine comparability
between a biosimilar and originator
product, developers use state-of-the-art
orthogonal analytical techniques (11,
54). In some cases, surrogate bioassays
can be used to confirm the absence of
changes in HOS introduced by the new
manufacturing process of biosimilars.
Changes in such characteristics can
affect product potency (20, 21).
For biosimilars, a product’s
biological activity also can be an
indicator of stability and
manufacturing process consistency
across batches. In general, however,
bioassay variability precludes the
sensitivity of such assays to detect
minor changes in potency. So
although they are helpful, biological
and/or functional assays may not fill a
gap in analytical assay sensitivity to
detect minor conformational
differences between biosimilar
TMAbs and innovator products. It is
important to note that no analytical
test or combination for HOS has yet
been sufficiently validated for
analytical testing as a substitute for
clinical studies in the development of
a biosimilar TMAbs drug substance.
Without such validation, creating a
quantitative industry standard to
establish HOS similarity seems
premature. Instead, renewed
investment in developing predictable
and standardized HOS measurements
of validated reference materials is
envisioned as a path forward.
Looking Ahead
Next month, we will conclude this
two-part discussion by examining drug
product issues (especially related to
aggregation and excipients) and
considering the possibility of
calibrating analytical methods against
validated reference antibodies. We also
will present a case study before offering
some conclusions and perspectives.
Acknowledgments
This work was supported with funds and a
fellowship award from the Global Biological
Standards Institute (GBSI). We thank Dr.
Anthony Mire-Sluis at AstraZeneca (California)
and Dr. Hans Ebbers at the Utrecht Institute for
Pharmaceutical Sciences (The Netherland) for
their feedback and critical review. We are
grateful to University of Maryland and GBSI
staff for their support of this project. In
particular, we thank Mark Gibson from GBSI for
helping to put together early drafts of this
work. The content is solely the responsibility of
the authors and does not necessarily represent
the official views of the funding agency.
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Implications for Biosimilars. Nat. Rev. Drug
Simran J. Kaur is an ORISE research fellow
at the US Food and Drug Administration;
Darryl Sampey is president and CEO of
BioFactura, Inc. (Frederick MD); Lester W.
Schultheis is director of the Maryland
Regulatory Science Initiative, part of the
Fischell Department of Bioengineering at
the University of Maryland (College Park);
and Leonard P. Freedman is president of
the Global Biological Standards Institute
(Washington, DC). Corresponding author
William E. Bentley is co–principal-
investigator of the University of Maryland’s
Center of Excellence in Regulatory Science
and Innovation (M-CERSI) and distinguished
professor of the University of Maryland,
3122 Kim Engineering Building, Fischell
Department of Bioengineering, University of
Maryland, College Park, 20742; 1-301-405-
4321, fax 1-301-405-9953; bentley@umd.edu.
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