Review

Biosimilars: Harmonizing the Approval Guidelines

Sarfaraz K. Niazi

College of Pharmacy, University of Illinois, Chicago, IL 60612, USA; niazi@niazi.com; Tel.: +1-312-297-0000

Abstract: Biosimilar approval guidelines need rationalization and harmonization to remove the
inconsistencies and misconceptions to enable faster, safer, and more cost-effective biosimilars. This
paper proposes a platform for a model guideline based on the scientific evaluation of the regulatory
filings of the 130+ products approved in the US, UK, and EU and hundreds more in the WHO
member countries. Extensive literature survey of clinical data published and reported, including
Clinicaltrials.gov, a review of all current guidelines in the US, UK and EU, and WHO, and detailed
discussions with the FDA have confirmed that removing the animal and clinical efficacy testing and
fixing other minor approaches will enable the creation of a harmonized guideline that will best suit
an ICH designation.

Keywords: biosimilars; WHO; MHRA; BPCIA; FDA; EMA; ICH; approval guidelines; analytical
assessment; animal testing; efficacy testing; immunogenicity testing; biological drugs

1. Introduction
Biosimilars are recombinant DNA products that join DNA from different species and
subsequently insert the hybrid DNA into a host cell, often a bacterium or mammalian cell,
to express the target protein; this molecular chimera was first created by researchers from
UC San Francisco and Stanford in 1972 [1,2]. Stanley Cohen of Stanford and Herbert Boyer
Citation: Niazi, S.K. Biosimilars:
of UCSF received the US patent in 1980. Boyer co-founded Genentech, Inc. in 1976. The
Harmonizing the Approval
Guidelines. Biologics 2022, 2, 171–195.
Cohen-Boyer patents will eventually have more than 500 licensees to biotechnology and
https://doi.org/10.3390/
pharmaceutical companies and earn Stanford and UCSF more than USD 250 million in
biologics2030014 royalties. These patents have now expired [3,4].
On 26 July 1974, ten researchers, including six future Nobel Laureates (James Watson,
Academic Editor:
Paul Berg, Stanley Cohen, David Baltimore, Ronald Davis, and Daniel Nathans), wrote
Vasso Apostolopoulos
a letter to the most reputable journal Science [5] to urge the NIH to regulate the use of
Received: 12 May 2022 recombinant DNA technology and urged scientists to halt recombinant DNA experiments
Accepted: 22 July 2022 until they better understood whether the technique is safe. These concerns eventually led
Published: 28 July 2022 to the 1975 Asilomar Conference [6], where one hundred prominent scientists gathered to
discuss the safety of manipulating DNA from different species. The meeting resulted in a
Publisher’s Note: MDPI stays neutral
set of NIH guidelines in 1976 that has been revised several times since then [7].
with regard to jurisdictional claims in
published maps and institutional affil-
On 7 July 2022, the author published his letter in the same Science magazine that had
iations.
carried the earlier warning, suggesting a ban on the animal testing of copies of approved
recombinant DNA products, the biosimilars [8]. The US FDA is telling us not to test even
new recombinant products in animals, except for carcinogenicity assessment [9]. Efforts are
underway to forbid animal testing at the US Congressional level to remove the possibility
Copyright: © 2022 by the author. of animal testing being used to justify analytical dissimilarity.
Licensee MDPI, Basel, Switzerland. Biosimilars include monoclonal antibodies, cytokines, growth factors, enzymes, im-
This article is an open access article munomodulators, and thrombolytics, proteins extracted from animals or microorganisms,
distributed under the terms and including recombinant versions of these products (except clotting factors), and other non-
conditions of the Creative Commons vaccine therapeutic immunotherapies. Billions of patients receiving biosimilars have shown
Attribution (CC BY) license (https:// therapeutic equivalence [10–21]. None of these products have shown adverse events more
creativecommons.org/licenses/by/ than the reference product [22–28], including immunogenicity responses. It is estimated
4.0/).

Biologics 2022, 2, 171–195. https://doi.org/10.3390/biologics2030014 https://www.mdpi.com/journal/biologics

Biologics 2022, 2 172

that the cumulative exposure to EU-approved biosimilars was more than two billion pa-
tient treatment days in 2020 [29], with no adverse event reporting or withdrawal from the
market due to safety reasons and no biosimilar-specific adverse effects have been added to
the product information [29,30]. Such an impeccable record of safety and efficacy that is
better than the record for chemical drugs needs serious consideration about the regulatory
guidelines to assure that we are not wasting resources and committing unethical practices.
The fast growth of these products has brought over 250 FDA-approved peptides and
therapeutic protein products [31]. As the patents of these products expire, the need for
cost-effective copies, the biosimilars, is accelerated.
The EMA introduced the first biosimilar guideline and approved the first product in
2006 [32]. The EU currently lists 95 centrally approved biosimilars [33]. The FDA brought its
guidelines in 2009 [34] and has 37 products approved [35]. Both agencies have made public
the details of the regulatory submissions of biosimilars. As of April 2022, 86 European
Public Assessment Reports (EPAR) were available [36]. The FDA provides access to these
data through its website portal on AccessData [37]. No other regulatory agency makes this
information accessible. The WHO is not a regulatory agency; it only provides scientific
advice to its 194 member countries.
As the end of the Brexit transition period approached last year, the MHRA released
draft guidance for consultation that was finalized on 14 May 2022 [38]. This guideline will
likely change how biosimilars are approved and concur with most of the suggestions made
in this article.
Now with 17 years of the use of biosimilars, the safety and efficacy of biosimilars
have been fully validated, with no recall; the same holds for all clinical trials conducted,
including testing after switching and alternating, as suggested in the US. Thus far, no
clinical efficacy study has failed to meet the acceptance criteria.
Biosimilars would have been treated like generic products if it was possible to declare
them chemically equivalent. Thus, the backbone of the approval of biosimilars is their
analytical assessment in a side-by-side comparison with the reference product. Recent
advances in analytical sciences now allow more stringent evaluation, making all other tests
less sensitive in identifying clinically relevant functional attributes.
After a biosimilar product meets the analytical similarity criteria, it is generally put
through animal testing, despite the knowledge that the peculiar mechanism of action of
biological products involves mainly receptor binding, which is not possible because of the
lack of these receptors in most animal species.
After animal testing comes to the pharmacokinetic and pharmacodynamic profil-
ing as an extension of the analytical assessment—essentially, to test how the body “sees”
the molecule and how the molecule “sees” the body. These studies also establish bioe-
quivalence and other pharmacokinetic parameters that may be able to tell the differences
in the rate and extent of receptor binding. Recently, the author suggested novel pro-
tocols with narrow inclusion criteria and a two-dose, cross-over study to reduce the
study cost. The pharmacodynamic comparisons are also made in the same study, well as
immunogenicity responses.
The last stage of testing involves clinical safety and efficacy testing. Except for a small
number, all biosimilar products approved conducted extensive clinical efficacy testing;
none of these studies failed due to poor sensitivity, as observed in the case of animal testing.
Therefore, it is proposed to disallow such studies for these studies for the same reason as
suggested for animal testing—disallow approval of biosimilars based on fallacious clinical
efficacy testing.
A recent report from Mckinsey & Co. states [39] that “the biosimilars industry needs
to reduce its costs, particularly in drug development, to preserve its sustainability. A
typical biosimilar today costs between USD 100 million and USD 300 million to develop,
with clinical trials accounting for more than half of the budget. If biosimilars are to
deliver the promise of making biological drugs accessible, these cost barriers must come
down exponentially.
Biologics 2022, 2 173

This paper presents a scientific and rational plan to harmonize the approval guidelines
by removing irrelevant testing and resolving regulatory misconceptions to allow global
acceptance of approved biosimilars.

2. Regulatory Background
Harmonizing biosimilar approval guidelines requires a clear understanding of the
differences in the existing guidelines and their roots of inclusion. There is only one exam-
ple of harmonization, albeit partial—the International Conference for Harmonization of
Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH), which
was established in 1990. In 2015, the organization and operations were renewed (this was
called “ICH reform”) as a legal entity under Swiss law, and its name was changed to the
International “Council” for Harmonization of Technical Requirements for Pharmaceuticals
for Human Use (ICH) [40]. While the ICH guidelines are now widely accepted, they are
limited to specific technical issues such as testing methods and qualification of manufac-
turing operations. The ICH guidelines do not recommend any approval process even
among the three original members—US, EU, and Japan. After Brexit, the MHRA applied
for full membership to ICH since it was engaged as a member of the EU in the past; this
membership was approved on 16 June 2022.
It is the goal of this paper to suggest a similar harmonization to the ICH, without the
limitations found in the WHO guidelines that too suggest a harmonized approach among
its 194 countries but come short in scientific merit [41,42]. A harmonized guideline will
be based only on scientific yet rational suggestions to enable a cost-effective development
and cost reduction without compromising safety and efficacy. This harmonization will
also allow global acceptance of registration, allowing companies to sell their products at
substantially reduced prices because of the wider distribution possibility.

2.1. The US Scene

There are over 100 biosimilar programs enrolled with the FDA [43]. To expedite the
approval process, the FDA has taken several significant steps.
The FDA has created two new guidelines, the extension of the Q&A presentations [44]
and the third revised draft guidance [45] titled “New and Revised Draft Q&As on Biosimilar
Development and the BPCI Act”. The details refer to fulfilling pediatric assessment or PREA
requirements, post-approval filing, and asserting that the 351(k) cannot have a different
route or dosage form. However, the strength issue was delayed, adding new indications
and orphan exclusivity. The FDA also updated The Purple Book FAQ section [46].
FDA has also published new fact sheets to provide additional educational materials
on biosimilar and interchangeable products and the biosimilar regulatory review and
approval process.
The BPCIA states [47] that the “Secretary may determine, in the Secretary’s discretion,
that an element described in clause (i) (I) [the biosimilar testing] is unnecessary in an
application submitted under this subsection”. The FDA has subtly implemented this
change in its new biosimilar guidance. However, unlike the EMA, MHRA, or WHO, the
FDA is bound by the BPCIA legislation that states that “an application submitted under this
subsection shall include information demonstrating that the biological product is biosimilar
to a reference product based upon data derived from analytical studies, animal studies, and
clinical studies”. The new education material includes the phrase “in addition to analytical
studies, other studies that may be needed”, not shall be, as stated in the BPCIA.
The FDA posts details of its approval of biosimilar products. However, a biosimilar
developer may object and secure under the Freedom of Information Act [48].

2.2. The EMA Scene

In 2001, much of the EU’s directive-based legislation concerning the regulation of
medicines was codified as Directive 2001/83/EC. The EMA has issued concept papers,
draft guidance, and public scientific workshops. The EMA’s Committee for Medicinal
Biologics 2022, 2 174

Products for Human Use (CHMP) has also issued product class-specific guidance such as
recombinant erythropoietin, granulocyte-colony stimulating factor, recombinant human
soluble insulin, low-molecular-weight heparins, somatropin, and recombinant interferon
alfa [49]. However, EMA has announced that they intend not to issue more specific
biosimilar guidelines but instead prefer to give tailored advice on a case-by-case basis.
This change in the EMA policy came from the FDA, and such guidelines can misdirect the
development of biosimilars.
Patents are not a significant issue in the EMA filing; the litigation is left to the claiming
parties. The patent laws in the EU are also different. The exclusivity for biological drugs is
ten years in the EU and 12 years in the US, giving the EU filings at least a two-year head
start. However, the ten years of exclusivity for patents and other exclusivity rights can last
longer than ten years after market approval. In the EU, process patents are rarely awarded,
reducing the significant barrier experienced by US filings, where the patent dance involves
the product and a multitude of process patents. The differences in the patent laws between
the US and the EU significantly impact the speed and scope of introducing biosimilars.
This topic is of interest to determine whether a harmonized guideline should include the
intellectual property issue, as elaborated later in this paper [50].
EMA guidelines and the decision-making of the EMA in approving biosimilars have
evolved significantly; the EMA is now promoting removing animal testing, though it is
not yet been made clear. In addition, like the FDA, EMA has recently begun approving
biosimilars without requiring clinical efficacy testing.

2.3. The WHO Scene

The World Health Organization (WHO) is not a regulatory authority, but it is mandated
to support regulatory authorities in its 194 Member States. The WHO guidelines on
evaluating biosimilars [51] provide suggestions to National Regulatory Agencies (NRAs)
principles for approving biosimilars.
In 2019, the WHO Expert Committee on Biological Standardization (ECBS) considered
that a more tailored and potentially reduced clinical data package might be acceptable in
cases where the available scientific evidence supported this. In addition, the committee
endorsed the review of current scientific evidence to consider updating the Guidelines
to provide more flexibility and clarity. Thus, the WHO reviewed scientific evidence and
experience to identify issues/cases for further reducing non-clinical and clinical data. The
progress was reported to the committee in 2020 (72nd and 73rd report) [52]. It has resulted
in additional suggestions on evaluating biosimilar monoclonal antibodies (mAbs) and an
expanded Q&A document.
In April 2022, the WHO published [53] a revised guideline based on the 22 comments
received. While the newest guideline and suggestions made by the WHO represent the
views of global regulatory agencies, it still falls short of establishing a rational scientific
platform. Listed below are some of the notable shortcomings in the WHO understanding
that should not be made part of a harmonized guideline:
• The WHO states that “the clinical data should be generated using the biosimilar
product derived from the final manufacturing process, reflecting the product for which
authorization is being sought. Any deviation from this recommendation must be
justified, and additional data may be required. For changes in the manufacturing
process, relevant guidelines like the ICHQ5E should be followed”. However, the
ICH comparability guideline applies only to the changes in the manufacturing of
a biotechnology product that has already been approved and thus requires testing
the product before and after the change, not with the reference product. To avoid
confusion, the FDA has made a strong point by labeling these studies as “analytical
assessments,” not even analytical comparisons.
• In its earlier guidelines, the WHO had indicated no need for any statistical modeling
of the critical quality attribute comparisons. The recent draft suggests using statistical
modeling but warns about the risks of employing statistical tests on limited samples
Biologics 2022, 2 175

(false-positive and false-negative conclusions). This reluctance of the WHO to propose

solid statistical modeling has resulted in many agencies requiring only 3–4 lots [54]
for testing. It is well understood that a larger number of lots are required before the
statistical modeling can be initiated. The WHO also states that the most frequently
applied overall similarity criteria require that a certain percentage of the biosimilar
batches (usually between 90% and 100%) fall within the similarity range. Given that in
an equivalence range, 90% of biosimilar lots must fall within three standard deviations
for the reference product. This means that only one lot out of ten can fall outside the
range, but if there are less than ten lots tested, the analysis becomes moot.
• For efficacy studies, the WHO allows using a non-inferiority model discouraged by
the FDA and EMA as inappropriate to consider higher efficacy leading to higher
safety issues.
• The WHO suggests that the chosen reference product must have been marketed for
a “suitable period” with proven quality, safety, and efficacy to serve the reference
product. No suitable period is defined, and the advice has led to distrust in the safety
of biological drugs approved under stringent regulatory compliance. While there is a
12-year restriction in the US and ten years in the EU, the WHO member agencies do
not have to comply with this restriction. The WHO statement has caused great damage
to the adoption of biosimilars in developing countries, and it must be removed.
• The WHO suggests that a biosimilar developer may use one source of reference
product for analytical testing and another for clinical testing. This argument is illogical;
all testing should be performed using the same reference product derived from the
same manufacturing source and bearing the same approval designation.
• The WHO maintains its position, despite many criticisms, that regional agencies can
decide the labeling and prescribing information. This is not only improper, but it
is also unethical, giving the regulatory agencies to modify the safety and efficacy
disclosures. The FDA has provided details of how the prescribing information should
be developed; this should be followed by the WHO.
• According to the WHO, if a comparison reveals differences in product-related sub-
stances and impurities between the biosimilar and the reference product, the impact
of the differences on the clinical performance of the drug product (including its bio-
logical activity) should be evaluated. This is the most misguiding advice. A reference
product had been thoroughly tested with its impurity profile for safety and efficacy.
Unmatched impurities cannot be validated by any means, including animal testing or
clinical efficacy testing. The quantity of matched impurities can vary, within certain
limits, as they can only bring a change in efficacy that is not likely to be significant.
This argument extends to process-related impurities as well. The process-related
impurities can be adjusted; thus, there is little rationale for qualifying an impurity not
present in the reference product. If a biosimilar product production can remove these
uncertainties, it should, regardless of their assumed risk.
• In the past, the WHO had given little importance to accelerated or stress condition
testing; this is now changed to follow the rationale that these testing are meant to be
part of the analytical assessment.
• The WHO statement, “It is up to the manufacturer to justify the relevance of the
established similarity ranges and criteria”, is inappropriate. These determinations
should be based on scientific principles, not individual agency preferences. This
advice from the WHO has resulted in the NRAs adopting irrational test limits without
justification.
• The WHO statement, “Nevertheless, any quality attributes not fulfilling the estab-
lished similarity criteria should be considered a potential signal for non-similarity
and assessed for possible impact on clinical safety and efficacy”, invites developers
to seek waivers based on animal or clinical testing. Here, the WHO goes back to
the assumption that differences in analytical similarity can be justified through any
non-clinical or clinical study.
Biologics 2022, 2 176

• WHO states, “Based on the totality of quality and nonclinical in vitro data available
and the extent to which there is residual uncertainty about the similarity of a biosimilar
and its reference product, it is at the discretion of the involved NRA to waive or not to
waive a requirement for additional nonclinical in vivo animal studies”. This statement
is misleading as it has caused many agencies to develop extensive animal testing, such
as the Indian CDSCO [54], which suggests using several times the human dose to
establish safety. The WHO further states, “To address the residual uncertainties, the
use of conventional animal species and specific animal models (for example, transgenic
animals or transplant models) may be considered”. This is not sound scientific advice,
leaving an impression that it may be possible to resolve differences in analytical
similarity using tests without relevance.
• WHO suggests that local tolerance studies are not required unless excipients are
introduced for which there is little or no experience with the intended clinical route of
application. Biosimilars can have formulations different from the reference product,
and a tolerance study is required to evaluate the formulation. If a formulation includes
ingredients that have not been used before, this creates significant risk and cannot
be resolved.
• According to the WHO, “Clinical studies should be designed to demonstrate confir-
mative evidence of the similar clinical performance of the biosimilar and the refer-
ence product, and therefore need to use testing strategies that are sufficiently sensi-
tive to detect any clinically relevant differences between the products”. The testing
strategies are always the same, either a response on a clinical marker. Both are the
least sensitive to tell the difference compared to analytical assessment and clinical
pharmacology testing.
• Using reference products remains unclear with issues such as using a foreign reference
product instead of a domestic product if a suitable reference product is not licensed
locally. In this case, the NRA may accept a reference product that has been licensed in
another jurisdiction.
• If required by the legislation in place, the comparability of the local and foreign-
sourced versions of the product should be demonstrated by analytical “bridging”
studies and, where needed, complemented by additional PK/PD data. Here the WHO
allows precedence of any local regulations to overcome scientific arguments.
• The WHO statement, “It may also be prudent not to waive the efficacy and safety study
when the reference product has common or unpredictable serious adverse effects that
cannot be merely explained by exaggerated pharmacological action”, is based on the
wrong assumption that efficacy study can overcome any unusual effects.
• The WHO also allows the NRAs to develop their prescribing information, which leads
to abuse of biosimilar products. There must be a unified approach to creating the label.

2.4. The MHRA Scene

Since exiting from Brexit, the MHRA has revised its guidelines independent of the
EMA or ICH guidelines. As the end of the Brexit transition period approached last year,
the MHRA released draft guidance for consultation that was finalized on 14 May 2022 [38].
This guideline has taken a more clear and more definite approach to the issues of animal
testing and clinical efficacy evaluation.

2.5. The ROW Scene

Most countries follow the guidelines discussed above; however, the WHO members
are more inclined to follow them unless they are more affluent, such as Saudi Arabia,
where the FDA/EMA guidelines apply. Many countries treat biosimilars like generic
chemical products with no clinical testing. In most cases, clinical testing is suggested for a
fixed number of patients. Recently, the concept of biological API (active pharmaceutical
ingredient) has risen, where companies import the drug substance and finish it locally.
Biologics 2022, 2 177

3. Definitions
3.1. Terminology
The first step in harmonizing the regulatory guidelines requires uniformity in the
terminology used in the context of biosimilar products. The terminology used in describing
biosimilars and the testing requirements can make a difference in the scope of testing. For
example, the term “biogeneric” was coined in 2004, but it was refused by the FDA on legal
grounds adopting “biosimilars” instead [55]. The term “biosimilar” means biologically
similar products, not otherwise, as it would be concluded if we call them similar biologicals.
Table 1 shows the current designations.

Table 1. Designation of off-patent biologics across the globe as of 2022.

Terminology Country
WHO, Canada, China, Egypt, Ghana,
Biosimilars Indonesia, Iran, Jordan, Malaysia, Korea,
Singapore, Thailand, USA, and Zambia
Follow-on Biologics Japan, Brazil
Similar Biologics India, Peru
Similar Biologic Medicinal Product (SBMP) EU, Ukraine
Multisource Known Biological Products Cuba
Bioanalogue Russia

Another term that needs attention is “comparability”, frequently used in place of

“similarity” when comparing a biosimilar candidate’s structural or biological attributes
with its reference product. The confusion starts with the ICH Guideline ICHQ5E [56]
“Comparability of Biotechnological/Biological Products”, which is intended to qualify the
changes in the manufacturing process of an approved biotechnology product, where the
“comparability” is established between the products and before and after the change, not a
reference product. Statements such as “for changes in the manufacturing process, relevant
guidelines like the ICHQ5E should be followed” [52] by the WHO are misleading. It has
caused developers to compare the development lots with their commercial product lots.
The similarity assessment can only be made with the reference product; the “comparability”
comes into the picture after a product has been approved. The FDA has made it more
explicit by replacing “testing” with “assessment”.

3.2. Reference Product

What constitutes a reference product should be uniform—a product approved using a
full dossier in one of the developed country’s regulatory agencies. This concept is not new;
when generic drugs came into existence, the WHO concluded that “Comparator products
should be purchased from a well-regulated market within the jurisdiction of a stringent
regulatory authority (SRA). For WHO Medicines prequalification, an SRA is considered the
regulatory authority of a country officially participating in the International Conference on
Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human
Use (ICH) and a member of ICH before 23 October 2015, namely: the US Food and Drug
Administration, the European Commission and the Ministry of Health, Labor and Welfare
of Japan also represented by the Pharmaceuticals and Medical Devices Agency. Therefore,
the consensus that the reference biological product must be approved by one of the initial
members of the ICH should be acceptable.
The WHO statement, “if the reference product is not authorized locally, the NRA may
allow the use of a product licensed by an experienced NRA that follows the WHO or corre-
sponding regulatory standards”, creates disharmony. The WHO further suggests bridging
studies if the reference product is licensed locally but sourced from another jurisdiction.
Biologics 2022, 2 178

There is a legal glitch in the BPCIA (FDA) that requires a reference product to be
“licensed”, and biologicals are “licensed” only in the US. There are many cases where the
same product is licensed in the US and authorized in the EU wherein “essentially” the
same registration dossier is submitted for registration. The EU will not require a bridging
study; the FDA has recently required a PK bridging study in such situations. The bridging
studies, especially clinical PK/PD studies, have been criticized since they complicate the
global development of biosimilars. The bridging studies should not be conducted if the
reference comparator has been approved in any ICH jurisdiction and there is evidence in
the public domain that the reference product has been approved in both jurisdictions upon
some of the same phase III clinical data” [57]. It is doubtful that the EU- and US-sourced
reference products have meaningful differences [58].
Health Canada permits using a foreign-sourced reference product licensed in an ICH
country when it does not have a locally sourced product [59].
A foreign-sourced reference product can be used in clinical studies. This is the case
for the EU and US, where a biosimilar must always refer to a local reference product for
legal reasons. Still, clinical studies can be performed with a non-European Economic Area
(EEA)/non-US version of the reference product, provided this has been authorized by a
regulatory authority with similar scientific and regulatory standards. In this case, the FDA
guidelines require analytical and PK/PD “bridging” studies by default. In contrast, the
EMA and Health Canada guidelines require analytical bridging, but PK/PD bridging only
if analytical bridging alone is insufficient.
The MHRA UK states that the reference product is sourced from the EU with evidence
that the RP is licensed in the EU via the centralized, decentralized, or mutual recognition
procedures, providing confirmation that these are the same as the Great Britain RP [54].

3.3. Materials and Standards

In-house Primary Reference Material: An appropriately characterized material pre-
pared by the manufacturer from a representative lot(s) for biological assay and physico-
chemical testing of subsequent lots and against which in-house working reference material
is calibrated.
In-house Working Reference Material: A material prepared similarly to the primary
reference material established solely to assess and control subsequent lots for the individual
attribute in question. It is always calibrated against the in-house primary reference material.
Publicly available reference standards (e.g., Ph. Eur.) cannot be used as the reference
product for the demonstration of biosimilarity. However, using these standards is vital in
method qualification and standardization.

4. Expression System
The Current US and EU guidelines allow the use of any expression system; however, if
the expression system is not the same, at least to the extent it is described in the prescribing
information (type, not necessarily the subtype), there can be many systematic issues with
product quality that will require additional testing. In addition, in some instances, any
“residual uncertainty” cannot be removed. The most widely used non-human host cell
lines for recombinant expression are CHO, NS0, Sp2/0, HEK293, and PER.C6, BHK21),
E. coli, and S. cerevisiae.
The developers are also advised to select an expression system that is more steady
than productive; recently, very high-yielding cell lines have been developed, but when cell
systems are pushed to produce, they also end up producing variants. Since the cost of goods
of recombinant products is based on the carbon input, it may not significantly alter the cost.
For example, the WHO calculates that the cost of production of monoclonal antibodies
ranges from USD 95–200 per gram [60] given the current market price of 100–1000× the
COGS; developers are advised to base their selection on cell lines that will allow for
faster approval if they produce a consistent product. Furthermore, important are the
Biologics 2022, 2 179

considerations in the contamination of cell lines with antibiotics that should be avoided in
their design.

5. Formulation
Biosimilars are allowed to have a formulation different from the reference product
formulation. However, unless prevented by intellectual property, a formulation with the
same or fewer inactive ingredients is preferred, notwithstanding any minor differences
in the composition of ingredients. If a different formulation is used, it may contain new
excipients that may not have been tested for the specific drug substance. If it is not
used for the formulation of biological products of the same classification; all excipients
must be free of animal materials; The formulation of the biosimilar should be selected
considering state-of-the-art technology and does not need to be identical to that of the
reference product. Regardless of the formulation selected, the suitability of the proposed
formulation with regards to stability, compatibility (i.e., interaction with excipients, diluents,
and packaging materials), integrity, activity, and strength of the active substance should
be demonstrated. Suppose a different formulation and container/closure system to the
reference product is selected (including any material in contact with the medicinal product).
In that case, its potential impact on the efficacy and safety of the biosimilar should be
appropriately justified.
Biosimilar epoetin α was the first epoetin α product that demonstrated the risk of
neutralizing antibodies cross-reacting with endogenous erythropoietin, which has caused
pure red cell aplasia in patients treated with the reference product [61]. This led to the
discontinuation of the development of the product for subcutaneous administration until
the underlying problem (which was not related to the quality of the active substance itself
but tungsten leaching from the needle of the syringe) was eliminated. Thus, the licensed
biosimilar epoetin α products have not shown excess immunogenicity compared to the
reference product [62–65].
A novel excipient not used in the recombinant protein formulations should be avoided.
The use of excipient(s) in the proposed biosimilar product not used in the RP is not
encouraged from a biosimilarity perspective. However, changes that may benefit patients
(for example, reducing injection pain or stinging) are encouraged and should be carefully
considered. Where different excipient(s) are used, there could be instances where this
would be the first time that this route had used the excipient; a discussion should be
presented addressing the safety of that excipient by the route intended.
In most instances, the excipient(s) will be used by the route intended at similar amounts
to other products. If so, a discussion to establish this can be sufficient. However, suppose a
novel excipient or a novel route for an excipient is used in the proposed biosimilar product.
In that case, this should be justified and includes the possibility that results from new safety
studies are presented, if appropriate. As studies intended to characterize the safety of the
excipient, compliance with GLP is expected.

6. Release Specification
The first step in developing a biosimilar product is establishing the release specifi-
cation drawn from the analysis of the reference product. Characterization of a biological
product includes the determination of its physicochemical properties, biological activity,
immunochemical properties, purity, and impurities using qualified testing methods. Test-
ing a larger number of reference product lots is favorable to biosimilar developers, as it
enables the justification of ranges of specifications that are more rational. The test lots can
come from the lots used throughout the development process. However, at least one lot
tested must be the one used for the first clinical trial, the PK/PD study.
The manufacturing process of the reference product evolves through its lifecycle,
which may lead to detectable differences in some quality attributes. Such events could
occur during the development of a biosimilar product. They may result in development
according to a QTPP, which is no longer fully representative of the reference product
Biologics 2022, 2 180

available on the market. The ranges identified before and after the observed shift in quality
profile could normally be used to support the biosimilar comparability exercise at the
quality level, as either range represents the reference product. Quality attribute values
outside or between the range(s) determined for a quality attribute of the reference product
should be appropriately justified concerning their potential impact on safety and efficacy.
Many legacy attributes are independently established, such as sterility, invisible parti-
cles (a controversial issue with biosimilars to consider them as aggregates), protein content,
potency, and physical properties specific to the biosimilar product; however, these remain
controversial. For example, the commonly acceptable for having no more than 3% impurity
and no single impurity more than 1% should be acceptable unless these ranges are higher,
in which case, they must be justified. In addition, the impurities must not include any im-
purity not found in the reference product. Any concessions in this regard are the remnants
of the understanding of the chemical products, where the immunological consideration is
unimportant. Therefore, attempts to justify the safety of unmatched impurity are futile; it
is better to remove the unmatched impurity.

7. Analytical Assessment
Analytical assessment is the strongest element of establishing biosimilarity. With
newer analytical technologies, it is now a more vigorous exercise. Though the critical
quality attributes are well-established, and so are the tests necessary, the developers have
shown great discord in the choice of tests. For example, companies have submitted different
numbers of analytical studies for adalimumab—25 by Pfizer and 71 by Boehringer—to
achieve the same goal [66].
There is a disconnect between what constitutes orthogonal testing and what is du-
plicate testing. An orthogonal test is required if a validated or suitable test can provide
aberrant results. For example, an HPLC method to measure protein content can be an
orthogonal test to UV absorbance testing, but not another spectrometric test or another
HPLC method. The validated methods are required for release testing but not for analytical
assessment, and it is for this reason that side-by-side testing is needed and all testing at the
same time.
The burden of analytical assessment can be significantly reduced if it is limited to qual-
ity parameters other than those included in the release specification. Every analytical assess-
ment report of approved biosimilars uses release specification parameters in analytical as-
sessment. For example, protein content or potency tests are release specification attributes.
The WHO does not consider a need for comparative stability profiling of the biosimilar
candidate with the reference product. Because of differences in formulation, a biosimilar
will have its lifecycle. Any process change post-approval should follow the ICH Q5E
guideline. However, the stability of the biosimilar product should be determined according
to ICH Q5C. The applicant should demonstrate that the desired product (including product-
related substances) present in the finished product of the biosimilar is similar to that of the
reference product. In contrast, process-related impurities may differ between the originator
and biosimilar products, although these should be minimized. It is preferable to rely on
purification processes to remove these impurities.
Product-related attributes are generally not modified by changing such parameters as
upstream conditions; these are mostly driven by the expression system. Product-related
attributes can and should be optimized; for example, one way to remove an unmatched
impurity is to lose the yield and cut off the peak instead of justifying the impurity. The EU
and UK fully agree with this suggestion [38].
The process-related attributes are not tested for analytical assessment; they are part
of the release specification established by testing multiple lots of the reference product.
However, any legacy attribute such as protein content or potency need not be tested in
both instances.
Testing requires reference materials, and there have been many misconceptions about
the role of pharmacopeias. Product release is based on using in-house reference materials,
Biologics 2022, 2 181

not on standards and reference materials (e.g., from Ph. Eur., WHO) that can only be used
for method qualification and standardization.
An interesting example of disputes relates to the release of insulin products. The
United States Pharmacopeia (USP) mandates an animal-based assay in rabbits in its Chap-
ter “<121> Insulin Assays” (USP <121>) for the potency evaluation (biological activity) of
insulin and insulin analogs. As the bioidentity test is mandatory in the US, it is included in
the quality specification for insulin drug substances for the US market. Since physicochemi-
cal assays such as HPLC assays used to determine the content of human insulin and insulin
analogs are much more precise and accurate than the rabbit blood sugar test, most of the
Pharmacopeias (e.g., in Europe, Japan, and India) decided to forgo the testing in living
animals. Consequently, the EMA recommends that marketing authorization holders use
the chromatography method for insulin, while the FDA insists on the rabbit test [67,68].
The pharmacopeias general monographs include tests for sterility, endotoxins, micro-
bial limits, volume in the container, uniformity of dosage units, and acceptable particulate
matter. However, if provided in a monograph, the specification is not acceptable to FDA
and EMA; a side-by-testing must be established.
The EMA provides more comprehensive guidance divided into immunogenicity
testing, quality issues, clinical and non-clinical testing, pharmacokinetic modeling, and
guidance on changing the manufacturing process of recombinant drugs. In addition, the
product-specific guidelines of the EMA are of great value for biosimilar developers [69].
The European and British Pharmacopoeias have developed monographs of several
critical biological products defining quality attributes to establish release specifications.
The USP has stated that it will not develop monographs for a biologic unless there is
stakeholder consensus supporting its creation, including the support of the FDA [70]. The
FDA has discouraged the USP from creating biologics monographs to ensure that innovator
biologics makers do not use the monograph process to block biosimilar competition by
incorporating patented characteristics of their product that are not relevant to safety, purity,
or potency, thereby further impacting competition [71]. The FDA also stayed away from
creating product-specific monographs, unlike the EMA.

Statistical Modeling
Comparing quality attributes is key in evaluating biosimilars and manufacturing
process changes. Different statistical approaches are required, but there is no regulatory
consensus on a quantitative and scientifically justified definition and an underlying hy-
pothesis of statistically equivalent quality. Therefore, the comparisons must be made using
methods to calculate the operating characteristics for false acceptance and rejection rates of
a claim for statistically equivalent quality. These error rates should be as low as possible to
allow a meaningful application of a statistical approach in regulatory decision-making.
Statistical data modeling, whenever comparative testing is conducted, is highly con-
troversial. An earlier FDA guideline, “Statistical Approaches to Evaluate Analytical Simi-
larity”, which recommended a rigorous statistical approach for establishing similarity, was
withdrawn [72] and replaced with a new guideline [73] in response to many objections,
including a citizen petition [74]. The new guideline removed the controversial tier one
assessment of quality attributes.
Historically, the WHO had maintained that there is no need for any statistical exercises
to compare the data; the most recent WHO guideline states: To mitigate the risks inherent in
employing statistical tests on limited samples (false-positive and false-negative conclusions),
a comprehensive control strategy must be established for the biosimilar to ensure consistent
manufacturing” [51,52]. However, while the guideline supports the quality range approach,
it fails to suggest a minimum number of lots needed, as does the FDA guideline [75,76].
The EMA, which had been silent on statistical methods, has described these in detail
in its newest guideline that describes the critical approaches for testing biosimilars [76].
While it recommends the interval range approach, it fails to mention the number of lots
required and leaves it up to the developer. A statistical testing requires a minimum number
Biologics 2022, 2 182

of lots to be of any value. Only the FDA guideline suggests using at least 6–10 lots, apart
from offering to conform to the suitability of the number of lots required.
A basic understanding of data teaches us that the application of any model is based
on the nature of data; if it is normally distributed, then many tests can be applied. The first
consideration when applying statistical tests is the essential flexibility of the requirement
of “high similarity” to allow for differences if they are clinically meaningless. Statistics
may facilitate the detection of differences, e.g., in data distributions or ranges. Still, the
determination of whether these differences are clinically relevant is a scientific question that
cannot be addressed by a statistical approach alone. Here is a list of various approaches to
compare the two products used [76].
• Visual display. This is most suitable where spectra are produced; most important is
the peak locations. This is one of the most important tests as it applies to the critical
comparison of primary, secondary, and tertiary structures.
• MinMax: A MinMax range is defined by a sample’s lowest and highest values. The
MinMax test is accepted if the MinMax range of the test sample is within the Min-
Max range of the reference sample (minTest > minRef and maxTest < maxRef). The
MinMax is a conservative approach with a low false acceptance rate but a high false
rejection rate.
• 3Sigma: the 3Sigma range is calculated for the reference sample as (µref-3σref, µref +
3σref). The 3Sigma test is accepted if the MinMax range of the test sample is within
the 3Sigma range. The 3Sigma approach provides a more practical compromise of
error rates, further improving with a larger sample size.
• Tolerance interval (TI): The tolerance interval is calculated for the reference sample as
(µ − k*σref, µ + k*σref). The k-factor is calculated two-sided with a confidence level
of 0.9 and a proportion of the population covered by the tolerance interval of p = 0.99.
The tolerance interval test is accepted if the MinMax range of the test sample is within
the tolerance interval calculated for the reference sample. Tolerance interval testing is
only usable if the sample size is sufficiently large.
• Equivalence testing of means (EQT): A two one-sided t-tests’ (TOST) procedure is
used to test for equivalency of the means of the reference product and the test product.
The equivalence margin is δ = 1.5 SDref (standard deviation of the reference product
sample). The Type I error probability is controlled at level α = 0.05 for a conclusion of
equivalence. The test is accepted if the (1 − 2α)100% = 90% confidence interval for
the difference in the means is within (−δ, +δ). Equivalence testing has a high false
rejection rate and, with increasing sample size, a considerable false acceptance rate.
The method of defining the acceptance ranges of critical quality attributes is well
described in EMA and FDA quality guidelines. In particular, the FDA biosimilar guidelines
thoroughly explain the risk assessment of quality attributes, while EMA guidelines refer to
other guiding documents. In general, both FDA, Health Canada, and EMA highlight the im-
portance of state-of-the-art orthogonal analytical methods and in vitro functional/potency
tests in the characterization of biosimilars. The quality section of the WHO guidelines
needs to be updated to align with the current expectations for analytical characterization
and demonstration of biosimilarity.
The quality attributes where statistical modeling is applicable include purity profile,
aggregate profile, and function assay profile; glycosylation is better compared with equiva-
lence test; the equivalent testing of mean is least likely to be of any value as used in every
regulatory submission.
While the purpose of analytical assessment is to show the difference, it is highly
unlikely that a significant difference can be justified; the test limits proposed in all the
above tests are arbitrary; it is for this reason, analytical differences are not allowed since, in
some cases, a minor change can produce an adverse response. The analytical assessment
extends to clinical pharmacology testing, discussed below.
Biologics 2022, 2 183

8. Non-Clinical Testing
Non-clinical in vitro testing has brought many new possibilities for characterization,
and these are considered part of the analytical assessment.
Non-clinical in vivo studies remain unresolved; the FDA BPCIA states, “demonstrat-
ing similarity based on evidence from pre-clinical studies (including toxicity assessment)
is required”. In several submissions, the FDA has refused to consider multiple animal
pharmacology and toxicology studies; however, the FDA remains mute on waivers of these
studies, even though the FDA has suggested that animal testing is not needed for new
biological drugs [9]. One reason for this dichotomy is the language used in the legislation
describing how biosimilars should be approved.
According to EMA, “Non-clinical studies should be performed stepwise before initi-
ating clinical studies. If necessary, in vitro studies should be conducted first, followed by
in vivo. These studies should be designed to detect differences between the similar biologi-
cal product and the reference product” [77]. However, the EMA has recently supported the
in vivo non-clinical testing waiver. It is up to the developers to seek the waiver. However,
what is needed is a ban on these useless studies a suggested by the author [9,78].
The WHO states, “Non-clinical evaluation of a new biotherapeutic normally encom-
passes a broad spectrum of PD, PK and toxicological studies to validate the efficacy and
safety of an SBP and should be comparative. Non-clinical studies should be performed in a
stepwise manner before initiating clinical studies. If necessary, in vitro studies should be
conducted first, followed by in vivo. These studies should be designed to detect differences
between the similar biological product and the reference product”.
In deciding the rationale for non-clinical testing, the WHO states, “Based on the
totality of quality and nonclinical in vitro data available and the extent to which there is
residual uncertainty about the similarity of a biosimilar and its RP, it is at the discretion
of the involved NRA to waive or not to waive a requirement for additional nonclinical
in vivo animal studies”. The WHO may consider using conventional animal species and
specific animal models (for example, transgenic animals or transplant models) to address
the residual uncertainties.
The EMA guideline provides the concept of step-wise progression in non-clinical
testing and reduction of animal work following the 3 R principles according to Directive
2010/63/EU. Health Canada states that in vivo toxicological studies are generally not
needed. It is acknowledged that biotechnology-derived proteins may mediate in vivo
effects that cannot be fully elucidated by in vitro studies. Therefore, non-clinical evaluation
of in vivo studies may be necessary to provide complementary information, provided that
a relevant in vivo model about species or design is available [79].
According to the revised UK guideline: “No in vivo studies from animals are requested
as these are not relevant for showing comparability between a biosimilar candidate and its
RP: this includes pharmacodynamic studies, kinetic studies, and toxicity studies” [80].
According to the EU and UK, factors to be considered when the need for in vivo
non-clinical studies is evaluated include, but are not restricted to:
• Presence of potentially relevant quality attributes that have not been detected in the
reference product (e.g., new post-translational modification structures).
• Presence of potentially relevant quantitative differences in quality attributes between
the biosimilar and the reference product.
• Relevant differences in formulation, e.g., excipients not widely used for biotechnology-
derived proteins.
If product-inherent factors that impact PK and biodistribution, such as extensive gly-
cosylation, cannot sufficiently be characterized on quality and in vitro level, in vivo studies
may be necessary. If there is a need for additional in vivo information, the availability of
a relevant animal species or other relevant models (e.g., transgenic animals, transplant
models) should be considered. If a relevant in vivo animal model is not available, the
applicant may choose to proceed to human studies considering principles to mitigate any
potential risk.
Biologics 2022, 2 184

Testing in animals is an old routine for new drugs to avoid toxicity to humans. It
works well for chemical drugs because the reactive chemical groups can interact with
multiple tissues to produce a toxic response. However, biological drugs may not always
show a pharmacologic response in animal species; thus, the toxicity is an extension of
the pharmacological response for biological drugs. The primary mechanism of action of
biological drugs involves receptor binding. A pharmacological or toxicological response is
not expected if an animal species does not carry these receptors.
Another reason animal toxicology data are less relevant is how the testing is conducted.
Generally, animal testing protocols require administering a higher dose to induce a toxic
response; however, within this dose range, the responses are not expected to be linear,
making it impossible to differentiate between compared products that are supposed to be
the same. Nevertheless, despite this knowledge and expertise, animal testing is extensively
conducted for biosimilars, evidenced by the recent FDA and EMA filings.
Another controversial issue in animal studies is the use of non-human primates, the
only species that may have relevant receptors; it is frequently recommended to conduct PK
studies in a small number of animals, especially for monoclonal antibodies, as a measure
of their molecular structure rather than toxicity. According to the recent statements from
WHO [54], “based on regulatory experience gained to date in marketing authorization
applications for biosimilars, the need for additional in vivo animal studies would be
expected to represent a rare scenario”. However, the guidelines in India take a very
different view, stating, “Regarding the animal models to be used, the applicant should
provide the scientific justification for the choice of animal model(s) based on the data
available in scientific literature. However, toxicity studies need to be undertaken in rodent
or non-rodent species if the pharmacologically relevant animal species are unavailable and
appropriately justified”. This requirement was implemented because India requires at least
one animal toxicology study, and no studies are allowed on monkeys for religious reasons.
Human and animal cells, organoids, organs-on-chips, and in silico modeling are
alternatives to animal testing models, enabling us to create better and more predictive
scientific methods. In addition, to reflect changes in animal protection legislation, non-
clinical in vivo testing has been substituted by in vitro assays in the previous ten years.
These measures can help reduce animal use. They also align with the EMA’s Regulatory
Science Strategy for 2025, aiming to create a more adaptive regulatory framework that
promotes human and veterinary health [80].
Animal toxicological studies can be misleading if they rationalize discrepancies in
impurities, post-translational modifications, or antibody responses since an animal model
can justify these differences. For example, animal data were submitted in biosimilar appli-
cations [66] to substantiate such variability, but the FDA refused to accept the animal data.
The EMA and FDA have approved more than 130 products. None of them have failed
animal toxicological testing because they cannot, being least sensitive in detecting any
difference between a biosimilar candidate and its reference product. These observations and
conclusions are widely accepted as scientifically sound arguments, but among sponsors,
there is always fear that study results will be rejected eventually. This would cause a delay
in market access at a high cost, and therefore sponsors like to stay on the safe side by
overpowering their studies.
There is great awareness of the futility of the animal testing of biosimilars, but this
will soon become a moot point, as the US Senate is considering a bill to remove the animal
testing of biosimilars [81]. Section (bb) is amended from “(bb) animal studies (including
the assessment of toxicity” to “an assessment of toxicity (which may rely on, or consist of,
a study or studies described in item (aa) or (cc)); (aa) is analytical assessment and (cc) is
clinical testing. This bill sponsored by Senator Lujan of New Mexico is now on the table in
Senate and expected to be signed soon [81].
Other agencies will follow suit.
Biologics 2022, 2 185

9. Clinical Evaluation
The need for clinical efficacy, as described in the FDA guidance, has brought much
confusion and misunderstanding [44]:
“As a scientific matter, FDA expects a sponsor to conduct comparative human
PK and PD studies (if there is a relevant PD measure(s)) and a clinical immuno-
genicity assessment. In certain cases, the results of these studies may provide
adequate clinical data to support the conclusion that there are no clinically mean-
ingful differences between the proposed biosimilar product and the reference
product. However, if residual uncertainty about biosimilarity remains after con-
ducting these studies, an additional comparative clinical study or studies would
be needed to evaluate whether there are clinically meaningful differences between
the two products.”
First, “clinical” does not mean testing in patients, and it could well be an in silico phar-
macokinetic study, as stated in the FDA’s Biosimilars Action Plan [82]. However, without
defining the “residual uncertainty”, without clarifying what is meant by “clinical”, the
regulatory agencies and the developers have assumed it to mean clinical efficacy testing as
a requirement; hundreds of such studies have been conducted, and none of these studies
failed, just like the animal toxicology testing, they cannot fail.

10. Clinical Pharmacology

It is now well-established that demonstrating comparable pharmacokinetics is critical
in the successful development and approval of most biosimilars [83].
The US, Canadian, and EU guidelines require comparative PK and, if relevant, PD
studies between the candidate biosimilar and its reference product by default [84]. The
current WHO guidelines also recommend this approach because PK(PD) studies are sensi-
tive to detecting potential product-related differences in vivo. Scientific reports underline
the importance of well-performed and robust PK studies with proper power calculation
avoiding too optimistic calculation of the inter-individual variability. Such PK studies
may already provide sufficient data on safety, including immunogenicity. In any case,
EMA, Health Canada, and FDA guidelines request safety and immunogenicity data for all
clinical studies [85,86].
The FDA states, “Comparative human PK, PD, clinical immunogenicity, clinical safety,
and effectiveness are required. In addition, at least one study with equivalence design and
adequate power is required to evaluate any clinically meaningful difference between the
proposed biosimilar product and the reference product”.
The EMA states, “PK/PD studies, followed by clinical efficacy and safety studies,
confirmatory studies for demonstrating clinical biosimilar comparability are required. In
addition, at least one study with adequate power and equivalence design in a sensitive
population is required to detect potential differences concerning efficacy and safety”.
Clinical pharmacology comparisons comprise the most relevant testing to support
biosimilarity. First, however, misconceptions should be addressed; unlike the development
of new drugs, here the purpose is to compare [87,88], which means that limited inclusion
criteria can reduce the study size. For example, Sandoz’s first study, GP17-101, failed to
demonstrate PK biosimilarity between the GP2017 test molecule, the EU-sourced Humira,
and the EU- and US-sourced RPs. Therefore, the company reconsidered the study design
and conducted another trial, GP17-104. The revised design increased the sample size and
was restricted to male patients, randomized, and stratified by body weight. GP17-104 was
successful in demonstrating PK profile similarity [89].
The FDA Biosimilar Action Plan also recommends employing in silico methodologies
to compare biosimilars, including immunogenicity assessments. Since immunogenicity is
entirely structure-dependent, better analytical assessment techniques give greater confi-
dence in reducing or eliminating anti-drug antibody testing. In addition, impurities and
Biologics 2022, 2 186

aggregates induce extrinsic immunogenicity, which may be easily measured and compared
to a reference product as part of the analytical evaluation.

11. Clinical Safety and Efficacy Studies

The issue of clinical efficacy testing becomes more compliance-based in the US, where
the legislation has created two classes of biosimilars, one that is biosimilar with “clinically
meaningful difference with the reference product”, and another that is further tested in
multiple switching and alternating studies with the reference product, to receive the desig-
nation of the interchangeable biosimilar. Thus, the FDA has approved two interchangeable
products–insulin glargine and adalimumab. Hundreds of clinical studies on switching and
alternating have shown that there is no difference [90]. Decades of switching biological
drugs such as insulin have shown no reason to assume that switching will result in a
different response or safety issue. Moreover, alternating makes little sense, as there is no
rationale for returning to the switched product.
Apart from the waste of resources, the more significant loss is that this legislation
creates a lack of confidence in the biosimilar that is not interchangeable testing. This
difference will be used by larger companies, as many of the products not part of the
substitution program are conducting these studies. However, removing this category of
biosimilars will take a legislative action currently underway, but like other such changes, it
will take time.
FDA, Health Canada, and EMA guidelines provide some flexibility regarding the
phase III-type “confirmatory” clinical efficacy and safety studies if certain requirements are
met, especially the availability of PD markers that are relevant markers or even surrogates
for efficacy. The Health Canada guideline states, “The non-clinical and clinical programs
should be designed to complement the structural and functional studies and address
potential areas of residual uncertainty”. The FDA guideline presents points that must be
addressed if the confirmatory efficacy and safety study is considered dispensable.
However, for most products, especially biosimilar mAbs, resource-intensive, phase
III-type confirmatory studies with an equivalence design are still expected, a major hurdle
in the entry of biosimilars.
The US guidelines advise confirming efficacy and safety studies to be performed if
residual uncertainties remain after the previous development steps. However, this advice is
not rational. No animal or efficacy testing can resolve an issue at the analytical assessment
stage since no data are available to correlate the analytical differences with safety and
efficacy; moreover, the efficacy studies are the least sensitive. It is this perception that
needs to be removed. Justifying differences in the analytical properties can only lead to the
entry of more hazardous biosimilars. The dose responses for biological drugs are broad
and unknown; as a result, none of these studies have ever failed. Clinical testing requires
establishing a pre-specified margin of difference that is always arbitrary; this includes
bioequivalence testing and clinical efficacy responses, making all these studies much less
sensitive to identifying differences between a biosimilar product and its reference product.
Hundreds of clinical efficacy testing conducted show that none has failed.
The MHRA states: “although each biosimilar development needs to be evaluated on a
case-by-case basis, it is considered that, in most cases, a comparative efficacy trial may not
be necessary if sound scientific rationale supports this approach. Therefore, a well-argued
justification for the absence of an efficacy trial should be appended to CTD Module 1 of the
submitted application [38].
The immunogenicity of biological products is caused by the activation of B cells, which
generate T cells to express antibodies. However, anti-drug antibodies can be harmful to
healthy subjects in future studies. As a result, the FDA is researching new methods for deter-
mining immunogenic potential using tiny fragments of DNA-like molecules called aptamers
to test proteins and establish their exact structures to avoid the exorbitant costs of forecasting
which particular portions of such proteins will stimulate antibody production [91].
Biologics 2022, 2 187

Finally, if the immunogenicity profile differs but cannot impact the disposition profile,
the differences will be meaningless, as the FDA has acknowledged in its new guidance
on insulins [92].
The European Medicines Agency (EMA) has begun work on a pilot clinical trial
program aiming to advise how to decrease or eliminate clinical testing in biosimilar de-
velopment [93]. Comparative clinical trials are increasingly seen as sloppy techniques for
assessing biological agent similarity. As a result, the testing of biosimilars in patients is
more of a checkmark than a meaningful indication.
EMA states, “generally, clinical data aim to address slight differences shown at pre-
vious steps and to confirm the comparable clinical performance of the biosimilar and the
reference product”. Clinical data cannot be used to justify substantial differences in quality
attributes [94]. Therefore, the first argument relates to identifying “slight differences” or, as
the FDA labels it, “residual uncertainty”.
Thus far, no biosimilar product has been rejected based on clinical efficacy and safety
testing if they passed the rest of the testing. This means the products were biosimilar, or
the testing was too insensitive to detect any difference [95–102]. In both cases, this testing
becomes irrelevant. This concept of real-time testing is now also questioned by the FDA,
which stated that clinical efficacy testing is “broken” and that new digital technologies and
real-world evidence (RWE) are required, as outlined in the 21st Century Cure Act [103].
Biosimilars “may be approved based on PK and PD biomarker data without a com-
parative clinical study with efficacy endpoint(s)”, according to FDA guidance [102]. Using
PK and PD biomarker data in healthy participants or patients enables shorter and less
expensive clinical investigations and provides more sensitive testing than clinical efficacy
with endpoint(s), as demonstrated with filgrastim [103]. The FDA acknowledged this and
granted approval for filgrastim-aafi, filgrastim-sndz, pegfilgrastim-jmdb, pegfilgrastim-
cbqv, and epoetin alfa-epbx based solely on PD evaluation. Furthermore, the FDA identified
the features of PD biomarkers in its advice to assist sponsors in using PD biomarkers as
part of biosimilar development programs [104].
Another reason why the clinical efficacy testing of biosimilars can be fallacious is due
to the testing models used: equivalence or non-inferiority. In the equivalence testing mode,
we first determine the M1 or total efficacy value of the reference product—a highly variable
but available parameter; second, we select an acceptable range of difference, the M2, based
on a clinical judgment that usually cannot be definitive—at best, it is an arbitrary choice. As
a result, since both products are expected to be identical, equivalency studies are least likely
to fail. On the other hand, non-inferiority testing is contraindicated because a biosimilar
product showing a higher efficacy may also have more safety issues.
The study’s power weakness is a simpler explanation of why the clinical efficacy is
less sensitive. Table 2 lists the clinical efficacy testing reported on clinicaltrials.gov that
was completed; the study size is generally larger than the originator product used in its
placebo-controlled study. Still, the studies have very low power and never fail. It is not too
complex to understand this assertion. At a given study power and alpha value, the study
size depends on the anticipated difference; that works fine for placebo-controlled studies
but not when two products are tested that are supposed to be similar. The only purpose
these studies serve is a stereotype proof of concept requirement, even if it is an irrational
approach.
Many drugs, including anticancer drugs, require the homogeneity of the study popu-
lation, which is unlikely. Patients are inevitably exposed to multiple drugs and treatment
modalities; additionally, anticancer drugs have a low efficacy rate, further reducing the
statistical probability of identifying any difference. Oncology or other terminal illness
treatment efficacy studies face specific hurdles, such as enrolling a comparable group of
naive patients. Such investigations further fail due to the brief lifespans of patients, which
can disrupt the study design.
Biologics 2022, 2 188

Table 2. Number of subjects in clinical studies of biosimilars compared to the originator study
(www.clinicaltrials.gov (accessed on 1 July 2022)). (References [95–101] report the values for the
reference product).

Product Reference Product (N) Biosimilar Product (N)

Adalimumab (Humira) 70 200–860
Bevacizumab (Avastin) 307 450–763
Rituximab (Rituxan) 161 256–629
Trastuzumab 233 225–875
Ranibizumab (Lucentis) 37 312–712
Infliximab (Remicade) 63 199–584
Aflibercept (Eylea) 180 366–576

Another argument against clinical efficacy testing is the extrapolation of indications

allowed for the biosimilar product. If there are doubts about the safety or efficacy, they
should be tested in all indications, not just one selected by the developer, even where
the modes of action are the same. A good example is conducting a psoriasis study for
adalimumab approval instead of testing in psoriatic arthritis.
Several detailed analyses have confirmed that analytical assessments alone or combined
with human PK/PD studies can demonstrate comparable efficacy and safety [102–110].
Comparative in vitro functional testing confirms the similarity of active substances’
higher-order structures and functions. PK studies prove similar exposure from the final
(formulated) product and provide information on safety and immunogenicity. These are the
essential elements of biosimilar development. In contrast, the phase III-type efficacy and
safety studies are regarded as “confirmatory” [83,84,111], yet they hardly confirm anything
more than what has already been established. More than 130 biosimilars approved in the
EU and US show that none of the products was rejected or questioned based on clinical
efficacy studies—they all passed.
The observations listed above make the interchangeability status of biosimilars estab-
lished after multiple switching and alternating between the biosimilar product and the
reference product a moot point.
The author has submitted an amendment to the BPCIA, where it removes the sec-
tions related to “Interchangeability” and replaces “clinical data” in (aa) with “clinical
pharmacology data”.

12. Label
The FDA and EMA provide the detailed structure of prescribing information (label) in
biosimilars and agree that all risks associated with the reference product must be stated.
However, the WHO leaves it to the discretion of regional agencies [112].

13. Summary of Harmonized Guideline

It is unlikely that regulatory agencies will agree on a single guideline document. Still,
they can certainly agree on the basic principles that are scientifically driven to assure the
safety and efficacy of biosimilars without any unnecessary or illogical testing.
• A uniform terminology. Products labeled as “biosimilar” are “approved” using “analytical
assessment”. “Comparability” confuses the use of ICHQ5E; it is “comparative similarity”.
• The reference product should be the first approved as a new drug in the US, EU, UK,
or Japan. The test samples must be secured from the source country, not from any
other country where the same product is distributed unless the product is approved
using the same dossier; no bridging study is required.
• The expression system type (e.g., mammalian, bacterial, yeast, etc.) used should
preferably be the same as used by the reference product, notwithstanding differences
in the type of expression species that may not be known. In addition, there should be
no antibiotic contamination.
• Must demonstrate that process is controlled and reproducible. Use ICH guidelines.
Biologics 2022, 2 189

• All excipients should be free of animal products, and no novel excipients should be
added to the formulation.
• Pharmacopeia test methods can be used for validating test methods only, not for com-
parative testing. The specifications in pharmacopeia should not be used to establish
biosimilar specifications.
• Product-specific monographs and the specifications suggested cannot be used to
support the claim of biosimilarity.
• If legally possible, the formulation should be the same as the reference product; or one
with fewer components.
• It should have the exact mode of action, same concentration, same dose, same route,
and same indications.
• Release specifications should be based on testing of reference lots for only non-legacy
attributes. Legacy attributes may use compendial specification. All test methods to
establish specifications must be validated. Tolerance intervals may be preferred to
establish specifications.
• Product-related attributes that are not included in the release specification should be
compared using a suitable, not necessarily a valid, method. Compendial test methods
can be used if available.
• Analytical assessment of data subject to statistical testing must be derived from at
least 9–10 lots of the reference and biosimilar products. The data must be normally
distributed. Where applicable, the 3Sigma approach is recommended. The visual
comparison is applied wherever the output is a non-quantified graphic display.
• Forced stability testing should be part of the analytical assessment. It should include
at least one lot at a commercial scale and manufactured under cGMP compliance that
will be used as a clinical lot.
• No unmatched impurities unless a safety profile is established for the same impurity,
not the same type. It is preferred to modify the downstream process to remove
unmatched impurities.
• Process-related impurities must be fixed with process optimization.
• No animal testing.
• Pharmacokinetic/pharmacodynamic testing in healthy subjects or patients at two dose
levels; one at the full dose and the other at half the dose. A parallel-group switching
in the second cycle allows for immunogenicity evaluation and cross-reacting antibody
evaluation. Use restricted inclusion criteria to reduce study size. A lower dose is likely
to be more sensitive in establishing similarity [87].
• No changes in the product are allowed after the PK/PD study has been conducted.
ICHQ5E does not apply to biosimilars until they are approved. The clinical lot must
be GMP and at scale.
• The label must conform to the reference product label; use the FDA format available.
Must include all indications and no new indications [112].
• Post-market safety reporting is required.

14. Conclusions
Regulatory guidelines should be harmonized and be based on the scientific rationale
to assure the safety and efficacy of biosimilars. Furthermore, necessary is the avoidance of
studies that might lead to justification of differences that may not be proper such as animal
and clinical efficacy testing to justify analytical differences. While the three major agencies
have revised the guidelines over time as the safety and efficacy of biosimilars have been
well-established, many discords remain that can be readily resolved [113–115].
It is suggested that a single ICH guideline title, “Establishing Biosimilarity of Re-
combinant Products”, will suffice as described above. Regional guidelines will be more
challenging to change due to legal and political rather than scientific reasons. Any product
approved under this new ICH guideline should be acceptable by all regulatory agencies if
it meets all local legal requirements. This will also reduce the burden on the WHO, which
Biologics 2022, 2 190

has struggled to create guidelines and failed due mainly to the democratic nature of the
process.
While the above recommendation will apply to all jurisdictions, the case of the US is
different, as the FDA has to follow the Congressional Act that describes how biosimilars
should be approved. The US Congress has brought several bills to expedite the adoption of
biosimilars [116], but none have addressed the core reason for the high price of biosimilars.
Senator Lujan of New Mexico presented a bill on 20 May 2022 to “allow manufacturers
and sponsors of a drug to use alternative testing methods to animal testing to investigate
the safety and effectiveness of a biological product”, as recommended by the author [117].
However, it will require removing all mention and references to “interchangeability, and
amending the section as shown below (underline is added word):
(k)(2)(i)(I)(cc)
“a clinical pharmacology study or studies (including the assessment of immunogenic-
ity and pharmacokinetics or pharmacodynamics) that are sufficient to demonstrate safety,
purity, and potency;”.
Delete (k)(2)(iii) (III)(B) and all references to “interchangeability”.
These changes, as submitted to the US Senate by the author, will limit the clinical
studies to only pharmacology studies and eliminate the two-class system of biosimilars.
A harmonized ICH guideline, and the changes to the US legislation, will turn the des-
tiny of biosimilars, bringing the perspective of affordable biosimilars affordable into reality.

Funding: This research received no external funding.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The author declares no conflict of interest.

References
1. Jackson, D.A.; Symons, R.H.; Berg, P. Biochemical method for inserting new genetic information into DNA of Simian Virus 40:
Circular SV40 DNA molecules containing lambda phage genes and the galactose operon of Escherichia coli. Proc. Natl. Acad. Sci.
USA 1972, 69, 2904–2909. [CrossRef] [PubMed]
2. Cohen, S.N.; Chang, A.C.; Boyer, H.W.; Helling, R.B. Construction of biologically functional bacterial plasmids in vitro. Proc. Natl.
Acad. Sci. USA 1973, 70, 3240–3244. [CrossRef] [PubMed]
3. Cohen, S.; Boyer, H. Process for Producing Biologically Functional Molecular Chimeras. U.S. Patent 423722A, 2 December 1980.
Available online: https://patents.google.com/patent/US4237224A/en (accessed on 8 June 2022).
4. Feldman, M.P.; Colaianni, A.; Liu, K. Lessons from the Commercialization of the Cohen-Boyer Patents: The Stanford University
Licensing Program. Handbook of Best Practices. In Intellectual Property Management in Health and Agricultural Innovation: A
Handbook of Best Practices; MIHR: Oxford, UK; PIPRA: Davis, CA, USA, 2007; Available online: https://maryannfeldman.web
.unc.edu/wp-content/uploads/sites/1774/2011/11/Feldman_Colaianni_Liu_2017_Cohen-Boyer-Patents-and-Licenses.pdf
(accessed on 8 June 2022).
5. Berg, P.; Baltimore, D.; Boyer, H.W.; Cohen, S.N.; Davis, R.W.; Hogness, D.S.; Zinder, N.D. Potential Biohazards of Recombinant
DNA Molecules. Science 1974, 185, 303. [CrossRef]
6. Berg, P. Asilomar 1975: DNA modification secured. Nature 2008, 455, 290–291. [CrossRef] [PubMed]
7. NIH. Guidelines for Research Involving Recombinant DNA Molecules. Available online: https://osp.od.nih.gov/wp-content/
uploads/NIH_Guidelines.pdf (accessed on 8 June 2022).
8. Niazi, S. End animal testing for biosimilar approvals. Science 2022, 377, 162–163. [CrossRef] [PubMed]
9. Avila, A.M.; Bebenek, I.; Bonzo, J.A.; Bourcier, T.; Bruno, K.L.D.; Carlson, D.B.; Dubinion, J.; Elayan, I.; Harrouk, W.; Lee, S.L.; et al.
An FDA/CDER perspective on nonclinical testing strategies: Classical toxicology approaches and new approach methodologies
(NAMs). Regul. Toxicol. Pharmacol. 2020, 114, 104662. [CrossRef]
10. Kurki, P.; Barry, S.; Bourges, I.; Tsintili, P.; Wolff-Holz, E. Safety, Immunogenicity and interchangeability of biosimilar monoclonal
antibodies and fusion proteins: A regulatory perspective. Drugs 2021, 81, 1881–1896. [CrossRef] [PubMed]
11. Weise, M.; Kurki, P.; Wolff-Holz, E.; Bielsky, M.C.; Schneider, C.K. Biosimilars: The science of extrapolation. Blood 2014, 124,
3191–3196. [CrossRef]
12. Doevendans, E.; Schellekens, H. Immunogenicity of innovative and biosimilar monoclonal antibodies. Antibodies 2019, 8, 21.
[CrossRef] [PubMed]
Biologics 2022, 2 191

13. Ebbers, H.C.; Pieper, B.; Issa, A.; Addison, J.; Freudensprung, U.; Rezk, M.F. Real-world evidence on etanercept biosimilar SB4 in
etanercept-naïve or switching patients: A systematic review. Rheumatol. Ther. 2019, 6, 317–338. [CrossRef]
14. Martelli, L.; Peyrin-Biroulet, L. Efficacy, safety and immunogenicity of biosimilars in inflammatory bowel diseases: A systematic
review. Curr. Med. Chem. 2019, 26, 270–279. [CrossRef] [PubMed]
15. Numan, S.; Faccin, F. Non-medical switching from originator tumor necrosis factor inhibitors to their biosimilars: Systematic
review of randomized controlled trials and real-world studies. Adv. Ther. 2018, 35, 1295–1332. [CrossRef]
16. Egeberg, A.; Ottosen, M.B.; Gniadecki, R.; Broesby-Olsen, S.; Dam, T.N.; Bryld, L.E.; Rasmussen, M.K.; Skov, L. Safety, efficacy and
drug survival of biologics and biosimilars for moderate-to-severe plaque psoriasis. Br. J. Dermatol. 2018, 178, 509–519. [CrossRef]
[PubMed]
17. Avila-Ribeiro, P.; Fiorino, G.; Danese, S. The experience with biosimilars of infliximab in inflammatory bowel disease. Curr. Pharm.
Des. 2017, 23, 6759–6769. [CrossRef]
18. Radin, M.; Sciascia, S.; Roccatello, D.; Cuadrado, M.J. Infliximab biosimilars in the treatment of inflammatory bowel diseases: A
systematic review. BioDrugs 2017, 31, 37–49. [CrossRef]
19. Moots, R.; Azevedo, V.; Coindreau, J.L.; Dörner, T.; Mahgoub, E.; Mysler, E.; Marshall, L. Switching between reference biologics
and biosimilars for the treatment of rheumatology, gastroenterology, and dermatology inflammatory conditions: Considerations
for the clinician. Curr. Rheumatol. Rep. 2017, 19, 37. [CrossRef]
20. Calleja-Hernández, M.Á.; Martínez-Sesmero, J.M.; Santiago-Josefat, B. Biosimilars of monoclonal antibodies in inflammatory
diseases and cancer: Current situation, challenges, and opportunities. Farm. Hosp. 2020, 44, 100–108. [CrossRef]
21. Esteva, F.J.; Lee, S.; Yu, S.; Kim, M.; Kim, N.; Stebbing, J. 24 months results from a double-blind, randomized phase III trial
comparing the efficacy and safety of neoadjuvant then adjuvant trastuzumab and its biosimilar candidate CT-P6 in HER2 positive
early breast cancer (EBC). Cancer Res. 2019, 79, 6–17. [CrossRef]
22. Stebbing, J.; Baranau, Y.; Baryash, V.; Manikhas, A.; Moiseyenko, V.; Dzagnidze, G.; Esteva, F.J. 3-year follow-up of a phase III trial
comparing the efficacy and safety of neoadjuvant and adjuvant trastuzumab and its biosimilar CT-P6 in HER2 positive early
breast cancer (EBC). European Society for Medical Oncology (ESMO) congress 2019. Ann. Oncol. 2019, 30, v63–v64. [CrossRef]
23. Stebbing, J.; Baranau, Y.; Baryash, V.; Manikhas, A.; Moiseyenko, V.; Dzagnidze, G.; Zhavrid, E.; Boliukh, D.; Stroyakovskii,
D.; Pikiel, J.; et al. CT-P6 compared with reference trastuzumab for HER2-positive breast cancer: A randomised, double-blind,
active-controlled, phase 3 equivalence trial. Lancet Oncol. 2017, 18, 917–928. [CrossRef]
24. Pivot, X.; Pegram, M.D.; Cortes, J.; Luftner, D.; Lyman, G.H.; Curigliano, G.; Yoon, Y.C. Final Survival Analysis of a Phase 3
Study Comparing SB3 (Trastuzumab Biosimilar) and Reference Trastuzumab in HER2-Positive Early or Locally Advanced Breast
Cancer. Available online: https://aacrjournals.org/cancerres/article/82/4_Supplement/P2-13-04/680824/Abstract-P2-13-04
-Final-survival-analysis-of-a (accessed on 12 July 2022).
25. Tony, H.P.; Krueger, K.; Cohen, S.B.; Schulze-Koops, H.; Kivitz, A.J.; Jeka, S.; Kollins, D. Brief report: Safety and immunogenicity
of rituximab biosimilar GP 2013 after switch from reference rituximab in patients with active rheumatoid arthritis. Arthritis Care
Res. 2019, 71, 88–94. [CrossRef]
26. Suh, C.H.; Yoo, D.H.; Berrocal Kasay, A.; Chalouhi El-Khouri, E.; Cons Molina, F.F.; Shesternya, P.; Lee, S.Y. Long-term efficacy
and safety of biosimilar CT-P10 versus innovator rituximab in rheumatoid arthritis: 48-week results from a randomized phase III
trial. BioDrugs 2019, 33, 79–91. [CrossRef] [PubMed]
27. Shim, S.C.; Božić-Majstorović, L.; Berrocal Kasay, A.; El-Khouri, E.C.; Irazoque-Palazuelos, F.; Cons Molina, F.F.; Yoo, D.H. Efficacy
and safety of switching from rituximab to biosimilar CT-P10 in rheumatoid arthritis: 72-week data from a randomized Phase 3
trial. Rheumatology 2019, 58, 2193–2202. [CrossRef] [PubMed]
28. Medicines for Europe. Biosimilar Medicines. The Total Clinical Experience with Biosimilar Medicines. Available online:
https://www.medicinesforeurope.com/wp-content/uploads/2020/12/BIOS5.pdf (accessed on 12 November 2021).
29. EMA. Biosimilars in the EU—Information Guide for Healthcare Professionals. Available online: https://www.ema.europa.eu/en
/documents/leaflet/biosimilars-eu-information-guide-healthcare-professionals_en.pdf (accessed on 12 November 2021).
30. Fischer, S.; Cohnen, S.; Klenske, E.; Schmitt, H.; Vitali, F.; Hirschmann, S.; Atreya, R. Long-term effectiveness, safety and
immunogenicity of the biosimilar SB2 in inflammatory bowel disease patients after switching from originator infliximab. Therap.
Adv. Gastroenterol. 2021, 14, 1756284820982802. [CrossRef] [PubMed]
31. Usmani, S.S.; Bedi, G.; Samuel, J.S.; Singh, S.; Kalra, S.; Kumar, P.; Ahuja, A.A.; Sharma, M.; Gautam, A.; Raghava, G.P.S. THPdb:
Database of FDA-approved peptide and protein therapeutics. PLoS ONE 2017, 12, e0181748. [CrossRef] [PubMed]
32. Daller, J. Biosimilars: A consideration of the regulations in the United States and European Union. Regul. Toxicol. Pharm. 2016, 76,
199–208. [CrossRef] [PubMed]
33. EMA. Centrally Approved Biosimilars. Available online: https://www.ema.europa.eu/en/medicines/field_ema_w
eb_categories%253Aname_field/Human/ema_group_types/ema_medicine/field_ema_med_status/authorised-36/em
a_medicine_types/field_ema_med_biosimilar/search_api_aggregation_ema_medicine_types/field_ema_med_biosimilar
(accessed on 8 June 2022).
34. US Congress. Title VII—Improving Access to Innovative Medical Therapies Subtitle A—Biologics Price Competition and
Innovation. Available online: https://www.fda.gov/media/78946/download (accessed on 23 March 2022).
35. FDA. Biosimilar Product Information. Available online: https://www.fda.gov/drugs/biosimilars/biosimilar-product-informati
on (accessed on 8 June 2022).
Biologics 2022, 2 192

36. European Medicines Agency. EPAR Biosimilars. Available online: https://www.ema.europa.eu/en/search/search/field_ema_w

eb_categories%253Aname_field/Human/ema_group_types/ema_medicine/search_api_aggregation (accessed on 8 June 2022).
37. FDA. Access Data. Available online: https://www.accessdata.fda.gov/scripts/cder/daf/ (accessed on 8 June 2022).
38. MHRA. Biosimilar Guidance. Available online: https://www.gov.uk/government/publications/guidance-on-the-licensing-of-
biosimilar-products/guidance-on-the-licensing-of-biosimilar-products (accessed on 8 June 2022).
39. Chen, Y.; Monnard, A.; Da Jorge Santos, S. An inflection Point for Biosimilars, McKinsey & Co. Available online: https:
//www.mckinsey.com/industries/life-sciences/our-insights/an-inflection-point-for-biosimilars (accessed on 8 June 2022).
40. ICH Harmonization. Available online: https://globalforum.diaglobal.org/issue/february-2021/ich-turns-30-perspectives-from
-japan/#:~{}:text=he%20International%20Conference%20for%20Harmonisation,the%20most%20resource%2Defficient%20man
ner (accessed on 1 July 2022).
41. Available online: https://www.centerforbiosimilars.com/view/whobiosimilarguidanceisbasedonweakscience (accessed on 8 June 2022).
42. Niazi, S. Opinion: One Step Forward, Half Step Back. Available online: https://www.centerforbiosimilars.com/view/opiniono
nestepforwardhalfastepbackforwhobiosimilarguidance (accessed on 8 June 2022).
43. FDA-TRACK: Center for Drug Evaluation & Research—Pre-Approval Safety Review—Biosimilars Dashboard. Available on-
line: https://www.fda.gov/about-fda/fda-track-agency-wide-program-performance/fda-track-center-drug-evaluation-res
earch-pre-approval-safety-review-biosimilars-dashboard (accessed on 23 March 2022).
44. FDA. Questions and Answers on Biosimilar Development and the BPCI Act Guidance for Industry. September 2021. Available
online: https://www.fda.gov/media/119258/download (accessed on 23 March 2022).
45. FDA. New and Revised Draft Q&As on Biosimilar Development and the BPCI Act (Revision 3) Guidance for Industry. September
2021. Available online: https://www.fda.gov/media/119278/download (accessed on 23 March 2022).
46. FDA. Purple Book. Available online: https://purplebooksearch.fda.gov/faqs#5 (accessed on 4 April 2022).
47. FDA BPCIA. Available online: https://www.fda.gov/media/779/download (accessed on 12 July 2022).
48. FDA. Freedom of Information Act. Available online: https://www.fda.gov/regulatory-information/freedom-information
(accessed on 23 March 2022).
49. European Medicines Agency. Human Regulatory. Biosimilars. Available online: https://www.ema.europa.eu/en/human-regul
atory/research-development/scientific-guidelines/multidisciplinary/multidisciplinary-biosimilar#-product-specific-biosimi
lar-guidelines-section (accessed on 23 March 2022).
50. Moorkens, E.; Vulto, A.G.; Huys, I. An overview of patents on therapeutic monoclonal antibodies in Europe: Are they a hurdle to
biosimilar market entry? MAbs 2020, 12, 1743517. [CrossRef]
51. WHO. Expert Committee on Biological Standardization. Annex 2. Guidelines on Evaluation of Similar Biotherapeutic Products (SBPs);
WHO Technical Report Series no 977; World Health Organization: Geneva, Switzerland, 2013; Available online: https://cdn.who.
int/media/docs/defaultsource/biologicals/trs_977_annex_2.pdf?sfvrsn=e2389a69_3&download=true (accessed on 8 June 2022).
52. WHO. 72nd and 73rd Report: WHO TRS N◦ 1030: 2020. Available online: https://www.who.int/publications/i/item/97892400
24373 (accessed on 8 June 2022).
53. WHO. Guidelines on Evaluation of Biosimilars. Replacement of Annex 2 of WHO Technical Report Series, No. 977. Available online:
https://www.who.int/publications/m/item/guidelines-on-evaluation-of-biosimilars (accessed on 8 June 2022).
54. CDSCO, India. Available online: https://cdsco.gov.in/opencms/resources/UploadCDSCOWeb/2018/UploadAlertsFiles/Biosi
milarGuideline2016.pdf (accessed on 8 June 2022).
55. Niazi, S. Handbook of Biogeneric Therapeutic Proteins: Regulatory, Manufacturing, Testing and Patent Issues; Taylor & Francis: Boca
Raton, FL, USA, 2002.
56. ICHQ5E Guideline. Available online: https://www.ema.europa.eu/en/documents/scientific-guideline/ich-q-5-e-comparability
-biotechnological/biological-products-step-5_en.pdf (accessed on 8 June 2022).
57. Webster, C.J.; Woollett, G.R. A ‘global reference’ comparator for biosimilar development. BioDrugs 2017, 31, 279–286. [CrossRef]
58. Tu, C.L.; Wang, Y.L.; Hu, T.M.; Hsu, L.F. Analysis of pharmacokinetic and pharmacodynamic parameters in EU-versus US-licensed
reference biological products: Are in vivo bridging studies justified for biosimilar development? BioDrugs 2019, 33, 437–446.
[CrossRef] [PubMed]
59. Health Canada. Information and Submission Requirements for Biosimilar Biologic Drugs. Available online: https:
//www.canada.ca/content/dam/hc-sc/migration/hc-sc/dhp-mps/alt_formats/pdf/brgtherap/applic-demande/guides/se
b-pbu/seb-pbu-2016-eng.pdf (accessed on 8 June 2022).
60. WHO. Call for Consultants on Antibodies. Available online: https://www.who.int/news-room/articles-detail/call-for-consult
ant-on-monoclonal-antibodies-for-infectious-diseases (accessed on 8 June 2022).
61. Casadevall, N.; Nataf, J.; Viron, B.; Kolta, A.; Kiladjian, J.; Martin-Dupont, P.; Michaud, P.; Papo, T.; Ugo, V.; Teyssandier, I.; et al.
Pure red-cell aplasia and anti-erythropoietin antibodies in patients treated with recombinant erythropoietin. N. Engl. J. Med. 2002,
346, 469–475. [CrossRef]
62. Haag-Weber, M.; Eckardt, K.U.; Hörl, W.H.; Roger, S.D.; Vetter, A.; Roth, K. Safety, immunogenicity and efficacy of subcutaneous
biosimilar epoetin-α (HX575) in non-dialysis patients with renal anemia: A multi-center, randomized, double-blind study. Clin.
Nephrol. 2012, 77, 8–17. [CrossRef] [PubMed]
63. Seidl, A.; Hainzl, O.; Richter, M.; Fischer, R.; Böhm, S.; Deutel, B.; Macdougall, I. Tungsten-induced denaturation and aggregation
of epoetin alfa during primary packaging as a cause of immunogenicity. Pharm. Res. 2012, 29, 1454–1467. [CrossRef]
Biologics 2022, 2 193

64. Wish, J.B.; Rocha, M.G.; Martin, N.E.; Reyes, C.R.D.; Fishbane, S.; Smith, M.T.; Nassar, G. Long-term safety of epoetin alfa-epbx
for the treatment of anemia in ESKD: Pooled analyses of randomized and open-label studies. Kidney Med. 2019, 1, 271–280.
[CrossRef]
65. Goldsmith, D.; Dellanna, F.; Schiestl, M.; Krendyukov, A.; Combe, C. Epoetin biosimilars in the treatment of renal anemia: What
have we learned from a decade of European experience? Clin. Drug Investig. 2018, 38, 481–490. [CrossRef] [PubMed]
66. Niazi, S. Analysis of FDA-Licensed Biosimilars: Time for a Paradigm Shift. AJMC, Center for Biosimilars. Available
online: https://www.centerforbiosimilars.com/view/analysis-of-fda-licensed-biosimilars-time-for-a-paradigm-shift
(accessed on 23 March 2022).
67. EMA. Insulin. Available online: https://www.ema.europa.eu/en/documents/scientific-guideline/guideline-non-clinical-clinic
al-development-similar-biological-medicinal-products-containing_en-0.pdf (accessed on 8 June 2022).
68. Hack, R. The Replacement of the Rabbit Blood Sugar Bioidentity Assay by an In Vitro Test for Batch Release of Insulin Glargine
Drug Substance. Available online: https://ec.europa.eu/environment/chemicals/lab_animals/3r/pdf/rudiger_hack.pdf
(accessed on 8 June 2022).
69. European Medicines Agency Biotechnology Products. Available online: https://www.ema.europa.eu/en/documents/scienti
fic-guideline/guideline-similar-biological-medicinal-products-containing-biotechnology-derived-proteins-active_en-0.pdf
(accessed on 23 March 2022).
70. USP. Statement on Monographs for Biologics. Available online: https://www.usp.org/news/statement-on-monographs-for-bio
logics (accessed on 23 March 2022).
71. FDA-USP Clash over Biologics Monographs. Available online: https://www.raps.org/news-and-articles/news-articles/2019/6/
fda-usp-clash-over-biologics-monographs (accessed on 4 April 2022).
72. Available online: https://www.fda.gov/drugs/drug-safety-and-availability/fda-withdraws-draft-guidance-industry-statist
ical-approaches-evaluate-analytical-similarity (accessed on 23 March 2022).
73. FDA. Development of Therapeutic Protein Biosimilars: Comparative Analytical Assessment and Other Quality-Related Consider-
ations Guidance for Industry. Available online: https://www.fda.gov/regulatory-information/search-fda-guidance-documents
/development-therapeutic-protein-biosimilars-comparative-analytical-assessment-and-other-quality (accessed on 23 March
2022).
74. Forbes Magazine. Scientist Invented A New Pathway To Approve Biosimilars, And The FDA Is Listening. Available on-
line: https://www.forbes.com/sites/nicolefisher/2018/07/25/one-mans-mission-to-fix-the-fdas-biosimilar-problem/?sh=1
843e1723808 (accessed on 23 March 2022).
75. EMA. Statistical Methodology for the Comparative Assessment of Quality Attributes in Drug Development. Available on-
line: https://www.ema.europa.eu/documents/scientific-guideline/reflection-paper-statistical-methodology-comparative-as
sessment-quality-attributes-drug-development_en.pdf (accessed on 8 June 2022).
76. Stangler, T.; Schiestl, M. Similarity assessment of quality attributes of biological medicines: The calculation of operating
characteristics to compare different statistical approaches. AAPS Open 2019, 5, 4. [CrossRef]
77. EMA. Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the Protection of Animals
Used for Scientific Purposes Text with EEA Relevance. European Medicines Agency. Directive 2010/63/EU. Available online:
http://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX:32010L0063 (accessed on 23 March 2022).
78. Niazi, S.K. Contributor No Animal Testing of Biosimilars—US Congress Begins Amendment to BPCIA. Available online: https://
www.centerforbiosimilars.com/view/contributor-no-animal-testing-of-biosimilars-us-congress-begins-amendment-to-bpcia
(accessed on 23 March 2022).
79. Moore, T.J.; Mouslim, M.C.; Blunt, J.L.; Alexander, G.C.; Shermock, K.M. Assessment of availability, clinical testing, and US Food
and Drug Administration review of biosimilar biologic products. JAMA Intern. Med. 2020, 181, 52–60. [CrossRef] [PubMed]
80. EMA. Regulatory Science to 2025. Available online: https://www.ema.europa.eu/en/documents/regulatory-procedural-guideli
ne/ema-regulatory-science-2025-strategic-reflection_en.pdf (accessed on 8 June 2022).
81. Senator Lujan Introduces Legislation to Reduce Animal Testing. 20 May 2022. Available online: https://www.lujan.senate.gov/n
ewsroom/press-releases/lujan-introduces-legislation-to-reduce-animal-testing/ (accessed on 8 June 2022).
82. FDA. Biosimilars Action Plan. Available online: https://www.fda.gov/media/114574/download (accessed on 10 July 2022).
83. FDA. Clinical Pharmacology Data to Support a Demonstration of Biosimilarity to a Reference Product. Available online:
https://www.fda.gov/media/88622/download (accessed on 23 February 2021).
84. EMA. Guideline on Similar Biological Medicinal Products Containing Biotechnology-derived Proteins as Active Substance:
Non-clinical and Clinical Issues. Revision 1. Available online: https://www.ema.europa.eu/en/documents/scientific
-guideline/guideline-similar-biological-medicinal-products-containing-biotechnology-derived-proteins-active_en-2.pdf
(accessed on 8 June 2022).
85. FDA. Immunogenicity of Protein-Based Therapeutics. June 2020. Available online: https://www.fda.gov/vaccines-blood-biologi
cs/biologics-research-projects/immunogenicity-protein-based-therapeutics (accessed on 23 March 2022).
86. EMA. Guideline on Immunogenicity Assessment of Therapeutic Proteins. Rev 1. Available online: https://www.ema.europa.e
u/en/documents/scientific-guideline/guideline-immunogenicity-assessment-therapeutic-proteins-revis12.11.ion-1_en.pdf
(accessed on 8 June 2022).
Biologics 2022, 2 194

87. Niazi, S. FDA Testimony. Available online: https://www.regulations.gov/document/FDA-2019-P-1236-0003 (accessed on 4

April 2022).
88. Gherghescu, I.; Delgado-Charro, M.B. The Biosimilar Landscape: An Overview of Regulatory Approvals by the EMA and FDA.
Pharmaceutics 2020, 13, 48. [CrossRef] [PubMed]
89. Bellinvia, S.; Fraser Cummings, J.R.; Ardern-Jones, M.R.; Edwards, C.J. Adalimumab biosimilars in Europe: An overview of the
clinical evidence. BioDrugs 2019, 33, 241–253. [CrossRef] [PubMed]
90. Druedahl, L.C.; Kälvemark Sporrong, S.; Minssen, T.; Hoogland, H.; De Bruin, M.L.; van de Weert, M.; Almarsdóttir, A.B.
Interchangeability of biosimilars: A study of expert views and visions regarding the science and substitution. PLoS ONE 2022, 17,
e0262537. [CrossRef]
91. FDA. Immunogenicity Assessment for Therapeutic Protein Products. Available online: https://www.fda.gov/media/85017/do
wnload (accessed on 12 July 2022).
92. FDA. Clinical Immunogenicity Considerations for Biosimilars and Interchangeable Insulin Products. Available online:
https://www.fda.gov/regulatory-information/search-fda-guidance-documents/clinical-immunogenicity-considerations-bi
osimilar-and-interchangeable-insulin-products (accessed on 23 March 2022).
93. EMA. Tailored Scientific Advice for Biosimilars Development. Available online: https://www.ema.europa.eu/en/documents/r
eport/tailored-scientific-advice-biosimilar-development-report-experience-pilot-2017-2020_en.pdf (accessed on 23 March 2022).
94. Biosimilars Clinical Testing Registered. Available online: https://clinicaltrials.gov/ct2/results?cond=&term=biosimilar&cntry
=&state=&city=&dist= (accessed on 23 March 2022).
95. FDA. Available online: https://www.accessdata.fda.gov/drugsatfda_docs/nda/2002/BLA_125057_S000_HUMIRA_MEDR_
P1.PDF (accessed on 23 March 2022).
96. FDA. Available online: http://www.fda.gov/downloads/Drugs/DevelopmentApprovalProcess/HowDrugsareDevelopeda
ndApproved/ApprovalApplications/TherapeuticBiologicApplications/UCM307496.pdf (accessed on 23 March 2022).
97. FDA. Available online: https://www.accessdata.fda.gov/drugsatfda_docs/nda/2006/125156s0000_LucentisTOC.cfm (accessed
on 23 March 2022).
98. FDA. Available online: https://www.accessdata.fda.gov/drugsatfda_docs/nda/2004/STN-125085_Avastin_medr_P1.pdf (ac-
cessed on 23 March 2022).
99. FDA. Available online: https://www.accessdata.fda.gov/scripts/cder/daf/index.cfm?event=BasicSearch.process (accessed on
23 March 2022).
100. FDA. Available online: https://www.accessdata.fda.gov/scripts/cder/daf/index.cfm?event=overview.process&ApplNo=10
3705 (accessed on 23 March 2022).
101. FDA. Available online: https://www.accessdata.fda.gov/drugsatfda_docs/nda/2011/125387s0000TOC.cfm (accessed on 23
March 2022).
102. FDA. 21st Century Cures Act. Available online: https://www.fda.gov/regulatory-information/selected-amendments-fdc-act/2
1st-century-cures-act (accessed on 23 March 2022).
103. Li, L.; Ma, L.; Schrieber, S.J.; Rahman, N.A.; Deisseroth, A.; Farrell, A.T.; Wang, Y.; Sinha, V.; Marathe, A. Quantitative relationship
between AUEC of absolute neutrophil count and duration of severe neutropenia for G-CSF in breast cancer patients. Clin.
Pharmacol. Ther. 2018, 104, 742–748. [CrossRef]
104. Li, J.; Florian, J.; Campbell, E.; Schrieber, S.J.; Bai, J.P.; Weaver, J.L.; Hyland, P.L.; Thway, T.M.; Matta, M.K.; Lankapalli, R.H.; et al.
Advancing Biosimilar Development Using Pharmacodynamic Biomarkers in Clinical Pharmacology Studies. Clin. Pharmacol.
Ther. 2020, 107, 40–42. [CrossRef] [PubMed]
105. González, C.P.V.; Muñoz, C.G. The controversy around technical standards for similar biotherapeutics: Barriers to access and
competition? Pharmacoepidemiol. Drug Saf. 2020, 29, 1518–1522. [CrossRef] [PubMed]
106. Frapaise, F.X. The end of phase 3 clinical trials in biosimilars development? BioDrugs 2018, 32, 319–324. [CrossRef] [PubMed]
107. McCamish, M.; Woollett, G. The continuum of comparability extends to biosimilarity: How much is enough and what clinical
data are necessary? Clin. Pharmacol. Ther. 2013, 93, 315–317. [CrossRef]
108. Liu, J.; Eris, T.; Li, C.; Cao, S.; Kuhns, S. Assessing analytical similarity of proposed Amgen biosimilar ABP 501 to adalimumab.
BioDrugs 2016, 2016, 321–338. [CrossRef] [PubMed]
109. McCamish, M.; Woollett, G. The state of the art in the development of biosimilars. Clin. Pharmacol Ther. 2012, 91, 405–417.
[CrossRef] [PubMed]
110. Christl, L. FDA’s Overview of the Regulatory Guidance for the Development and Approval of Biosimilar Products in the US.
Available online: https://www.fda.gov/files/drugs/published/FDA%E2%80%99s-Overview-of-the-Regulatory-Guidance-f
or-the-Development-and-Approval-of-Biosimilar-Products-in-the-US.pdf (accessed on 8 June 2022).
111. WHO. Guidelines on Evaluation of Biosimilars. Available online: https://cdn.who.int/media/docs/default-source/biologicals/
bs-documents-(ecbs)/annex-3---who-guidelines-on-evaluation-of-biosimilars_22-apr-2022.pdf?sfvrsn=e127cbf4_1download=
true (accessed on 12 July 2022).
112. FDA. Biosimilar Labeling Guide. Available online: https://www.fda.gov/files/drugs/published/Labeling-for-Biosimilar-Produ
cts-Guidance-for-Industry.pdf (accessed on 8 June 2022).
113. Schiestl, M.; Ranganna, G.; Watson, K.; Jung, B.; Roth, K.; Capsius, B.; Trieb, M.; Bias, P.; Maréchal-Jamil, J. The path towards a
tailored clinical biosimilar development. BioDrugs 2020, 34, 297–306. [CrossRef]
Biologics 2022, 2 195

114. Wolff-Holz, E.; Tiitso, K.; Vleminck, C.; Weise, M. Evolution of the EU biosimilar framework: Past and future. BioDrugs 2019, 33,
621–634. [CrossRef] [PubMed]
115. Van Aerts, L.A.; De Smet, K.; Reichmann, G.; van der Laan, J.W.; Schneider, C.K. Biosimilars entering the clinic without animal
studies, a paradigm shift in the European Union. MAbs 2014, 6, 1155–1162. [CrossRef] [PubMed]
116. Congressional Bills under Process or Approved on Biosimilars. Available online: https://insidehealthpolicy.com/search/biosim
ilar (accessed on 12 July 2022).
117. TAM22904 R6D. S.L.C. 117TH CONGRESS. 2D SESSION. S. Available online: https://www.lujan.senate.gov/wp-content/uplo
ads/2022/05/TAM22904.pdf (accessed on 8 June 2022).