establishing biosimilarity. 8–10
Critical quality attributes of a biologic have the
potential to affect its safety or efficacy through a
variety of mechanisms; thus, the definition of these
attributes is important for assuring the efficacy and
safety of the product. In the case of monoclonal
antibodies (mAbs), efficacy can be affected by
changing the antibody’s interaction with its target,
for example, due to conformation alterations or
chemical modifications of critical residues. These
attributes might alter the safety profile of the
therapeutic antibody, either by increasing potential
immunogenicity or by causing an increase in off-
target binding, for example.
A single quality attribute might affect multiple
functions, and the degree of impact can be molecule-
specific. Most therapeutic proteins undergo
glycosylation, which can influence the biological
activity of a protein. For example, glycosylation
can affect half-life by influencing the clearance of
a protein, and in the specific case of therapeutic
antibodies, Fc glycosylation plays a role in functions
such as antibody-dependent cellular cytotoxicity. 11–13
Reverse engineering
The manufacturing process of biosimilars tries
to ‘replicate’ that of the reference without full
knowledge of its different steps – considering that
it is proprietary – through detailed analyses of the
reference product and using a ‘reverse engineering’
strategy. Given the structural complexity and
variation of the molecules and the cell systems
where they are produced, reverse engineering may
result in significant differences in the production
of the biosimilar (for example, different cell line,
production and purification of the biosimilar from
the cell line). This poses significant challenges
to companies and their scientific teams in terms
of expertise and capabilities as well as time and
cost. However, because the focus of clinical trials
involving biosimilars is on the need to show similar
physicochemical and biological properties as well
as efficacy and safety versus the original molecule,
and not on elucidating the mechanism of action and
proof of concept, the regulatory paths to approval
are distinct and bypass some of these inherent
difficulties. 14
Comparability
Comparability studies need to be conducted to
demonstrate the similarity in terms of quality, safety
and efficacy, of the biosimilar and its approved
reference drug. 2 In addition, the comparability and
similarity of its biological activity is determined
using assays relevant to the modes of action of
the reference biologic in all potential indications
(Table 2). 15 The mechanisms of action of the drug
are assessed in all potential disease indications
to confirm that it performs adequately and
comparatively to the reference drug under clinical
conditions. This thorough assessment is designed
to show high similarity because all aspects of
the drug are evaluated with the most sensitive
and specific assays. Together these assays should
broadly cover the functional aspects of the drug,
even though some may not be considered essential
for the therapeutic mode of action. Because in
vitro assays can be more specific and sensitive
than studies in animals, they are paramount
in the non-clinical comparability exercise. This
entirety of evidence ensures that all accumulated
data (clinical and analytical) on the biosimilar
candidate are considered. This includes comparison
of the bioanalytical characteristics of batches of
reference product and biosimilar, and the effects on
the cellular and molecular mechanisms of action
relevant to the disease indications of the reference
drug. 16
Immunogenicity
For a biologic that is administered to an individual
more than once, alternating or switching between
the biosimilar and reference products should not
increase the risk of adverse events from using the
reference product without such alternation or
switching in terms of safety or diminished efficacy.
Similarly, the risk associated with alternating
or switching between biosimilars should not be
greater than the the risk of uninterrupted use of the
reference product. 17
Therefore, immunogenicity (that is, the ability of
the biosimilar to trigger an immune reaction) is a
critical factor when assessing biosimilarity and should
be evaluated in a risk-based manner. Most biologics
can induce immune responses, which, in many cases,
do not have clinically relevant consequences. Immune
responses can include the development of antidrug
antibodies or generation of neutralising antibodies
that might potentially eliminate activity. The most
severe scenario is the cross-reaction of antidrug
antibodies with an endogenous protein, thereby
eliminating its critical function and potentially causing
harm. The extent of immunogenicity can vary due to
changes in the manufacturing processes of the same
biosimilar, or any biologic (including reference drugs),
or among different manufacturers of biosimilars/
reference products. 18–21
Scientific and immunological reasons might
TABLE 2
Comparability testing for biosimilars
Quality attribute Demonstration of similarity
Protein structure and manufacturing quality Extensive laboratory analyses of molecular
characteristics (in multiple batches)
Pharmacokinetics, pharmacodynamics and
toxicity (in animals) In vitro and in vivo assays (in vivo studies
only if additional information is needed after in vitro
evaluation)
Pharmacokinetics, pharmacodynamics and
toxicity (in humans) Early pharmacology studies
Clinical efficacy and safety Pivotal clinical comparability trials, usually in the most
sensitive indication
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