Drug Metabolism and Pharmacokinetics 34 (2019) 64e70

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pharmacokinetics

Review

Biosimilarity assessment of biosimilar therapeutic monoclonal

antibodies
Akiko Ishii-Watabe a, *, Takashi Kuwabara b
a
Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, Japan
b
Yokohama University of Pharmacy, Japan

a r t i c l e i n f o a b s t r a c t

Article history: The concept of biosimilar was established in the early 2000s in EU. Currently, the regulatory framework
Received 29 October 2018 for biosimilar has also been established in the US, Japan, and other countries. As of 2018, biosimilars for
Received in revised form infliximab, adalimumab, rituximab, trastuzumab, and bevacizumab have been approved. During the
27 November 2018
development of a biosimilar, product quality should be evaluated and compared with those of the
Accepted 28 November 2018
Available online 7 December 2018
reference product extensively. Among the quality attributes of therapeutic antibodies, FcRn binding and
related structures are well known to affect the pharmacokinetic profile of the product. Other quality
attributes such as antigen binding, glycan structure, and isoelectric point are considered to have a po-
Keywords:
Biosimilar
tential impact on the pharmacokinetic profile of the product. Based on the high similarity of the quality
Therapeutic antibody attributes of the biosimilar to those of its reference product, comparative non-clinical and clinical studies
Comparability are conducted. Comparable pharmacokinetic profile of the biosimilar and the reference product is
Biosimilarity important for biosimilar evaluation. In this review, the basic concept of biosimilar development as well as
Pharmacokinetics pharmacokinetic data obtained via non-clinical and clinical studies of biosimilar therapeutic antibody is
Comparative study introduced, and future perspective is discussed.
© 2018 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. This is an open access article
under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction
A biosimilar is a biotechnological product comparable to an
already approved biotechnological product (hereinafter “reference
product”) in terms of quality, safety and efficacy. Namely, having
“comparable” quality, safety and efficacy is recognized as “bio-
similar” to the reference product. According to the US FDA, bio-
similar or biosimilarity means that “the biological product is highly
similar to the reference product notwithstanding minor differences
in clinically inactive components, and that “there are no clinically
meaningful differences between the biological product and the
reference product in terms of the safety, purity, and potency of the
product” [1]. The European regulatory agency EMA has a similar
definition, wherein a biosimilar is defined as a medicine which is
similar to a biological medicine that has already been authorized
[2]. In Japan, the regulatory pathway for biosimilar marketing
authorization application is assigned for “follow-on biologics” [3];
* Corresponding author. Division of Biological Chemistry and Biologicals, National
Institute of Health Sciences, 3-25-26, Tonomachi, Kawasaki-ku, Kawasaki, Kana-
gawa 210-9501, Japan.
E-mail address: watabe@nihs.go.jp (A. Ishii-Watabe).
however, the products have been called “biosimilar” recently. The
concept of biosimilar is widely accepted worldwide, although there
are slight differences in the regulatory framework among each
regulatory agency [4].
The characteristics of active pharmaceutical ingredient of a
biosimilar must be highly similar, in structure, physicochemical and
biological properties, to those of the reference product. The pos-
ology and route of administration of the biosimilar are the same as
those of the reference product. Deviations from the reference
product as regards pharmaceutical form, formulation, and excipi-
ents should be justified [2].
The biosimilar therapeutic monoclonal antibodies (mAbs)
approved in the EU, the US, and Japan are listed in Table 1. The first
approved biosimilar mAb was the infliximab biosimilar, an anti-
TNFa mAb that is used for treatment of rheumatoid arthritis, pso-
riatic arthritis, plaque psoriasis, Crohn's disease, ulcerative colitis,
ankylosing spondylitis, and other related diseases. The infliximab
biosimilar was approved in 2013 in EU. By September 2018, bio-
similar mAbs for adalimumab (anti-TNFa mAb), rituximab (anti-
CD20 mAb), bevacizumab (anti-VEGF mAb), and trastuzumab (anti-
HER2 mAb) were also approved. In addition, biosimilar for eta-
nercept, a TNF receptor-Fc fusion protein was approved. Currently,

https://doi.org/10.1016/j.dmpk.2018.11.004
1347-4367/© 2018 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).
 A. Ishii-Watabe, T. Kuwabara / Drug Metabolism and Pharmacokinetics 34 (2019) 64e70 65

Table 1
Biosimilar mAb and Fc-fusion products approved in the EU, the US, or Japan.

Innovator product Biosimilar Approved year

Nonproprietary namea Trade name Company EU US Japan

Remicade infliximab Inflectra Hospira 2013 - -

infliximab Remsima Celltrion 2013 - -
infliximab Flixabi Biogen 2016 - -
infliximab Zessly Sandoz 2018 - -
infliximab-dyyb Inflectra Pfizer/Hospira - 2016 -
infliximab-abda Renflexis Samsung Bioepis - 2017 -
infliximab-qbtx Ixifi Pfizer - 2017 -
infliximab BS1 Infliximab BS for I.V. infusion [NK] [CTH] Nippon Kayaku, Celltorion - - 2014
infliximab BS2 Infliximab BS for I.V. infusion [Ayumi] [Nichiiko] Ayumi, Nichiiko - - 2017
infliximab BS3 Infliximab BS for I.V. infusion [Pfizer] Pfizer - - 2018
Humira adalimumab Amjevita, Solymbic Amgen 2017 - -
adalimumab Cyltezo Boehringer Ingelheim 2017 - -
adalimumab Imraldi Samsung Bioepis 2017 - -
adalimumab Hefiya, Halimatoz Sandoz 2018 - -
adalimumab-atto Amjevita Amgen - 2016 -
adalimumab-adbm Cyltezo Boehringer Ingelheim - 2017 -
MabThera rituximab Truxima, Blitzima, Ritemvia, Rituzena Celltrion 2017 - -
rituximab Riximyo, Rixathon Sandoz 2017 - -
Rituxan rituximab BS1 Rituximab BS Intravenous Infusion [KHK] Kyowa Hakko Kirin - - 2017
Avastin bevacizumab Mvasi Amgen 2018 - -
bevacizumab-awwb Mvasi Amgen - 2017 -
Herceptin trastuzumab Ontruzant MSD 2017 - -
trastuzumab Herzuma Celltrion 2018 - -
trastuzumab Kanjinti Amgen 2018 - -
trastuzumab Trazimera Pfizer 2018 - -
trastuzumab-dkst Ogivri Mylan - 2017 -
trastuzumab BS1 Trastuzumab BS I.V. infusion [NK] [CTH] Nippon Kayaku, Celltrion - - 2018
trastuzumab BS2 Trastuzumab BS I.V. infusion [DAIICHI SANKYO] Daiichi Sankyo - - 2018
trastuzumab BS3 Trastuzumab BS I.V. infusion [Pfizer] Pfizer - - 2018
Enbrel etanercept Benepali Biogen 2017 - -
etanercept-szzs Erelzi Sandoz - 2016 -
etanercept BS1 Etanercept BS for S.C. Inj. [MA] Mochida - - 2018

e.g., Infliximab (Genetical Recombination) [Infliximab Biosimilar 1] is shown as Infliximab BS 1.

a
Nonproprietary name of biosimiars approved in Japan is abbreviated.

biosimilars for other mAbs such as ranibizumab and omalizumab
are being developed [5].
In the development of biosimilars, comparison of clinical phar-
macokinetic (PK) profile is an important issue. This review sum-
marizes the principle of biosimilar development, and introduces
the points to be considered in comparative quality, non-clinical,
and clinical studies, focusing mainly on comparison of PK profile
of biosimilars and their reference products.
2. Concept for biosimilar evaluation
The scientific principles for a biosimilar comparability assess-
ment to the reference product are based on those applied for
evaluation of the impact of a change in the manufacturing process
of a biotechnological product (as outlined in ICH Q5E guideline [6])
[2]. A biosimilar should be highly similar to the reference product
with regard to quality attributes including structure, physico-
chemical and biological properties. Any observed differences in
quality attributes should be justified by showing that there are no
clinically meaningful differences between the biosimilar and the
reference product in terms of safety and efficacy.
As the first step of biosimilar development, the biosimilar
company establishes the manufacturing process of the biosimilar
by itself, as that for the reference product is not disclosed. There-
fore, the manufacturing process of a biosimilar is different from
that of its reference product. Since quality of biotechnological
products can vary depending on the manufacturing methods (e.g.,
host cells, cell culture conditions and purification procedures), the
comparability assessment of the biosimilar with its reference
product is critical.
The comparability assessment of biosimilar to the reference
product is pursued by a stepwise approach. The data required for
approval of biosimilars in contrast to those for innovator products
(reference products) are shown in Fig. 1 [7]. The comparability
assessment consists of comparative quality studies, comparative
non-clinical studies, and comparative clinical studies between
biosimilar and its reference product. Because the quality attributes
of a biosimilar are not identical to those of the reference product,
some differences are usually detected in the comparative quality
studies. Considering the impact of such differences on safety and
efficacy of biosimilars, in vitro or in vivo non-clinical studies are
performed. Then, to ensure that these differences do not lead to any
clinically meaningful differences, comparative clinical studies are
performed. The biosimilar comparative clinical study is normally a
stepwise procedure that should begin with PK and, if feasible,
pharmacodynamic (PD) studies followed by clinical efficacy and
safety trial(s) or, in certain cases, confirmatory PK/PD studies for
demonstrating clinical biosimilar comparability [8]. In the absence
of surrogate markers for efficacy, it is usually necessary to
demonstrate comparable clinical efficacy of the biosimilar and the
reference product in adequately powered, randomized, parallel
group comparative clinical trial(s), preferably double-blinded, by
using efficacy endpoints [8].
3. Comparability assessment of therapeutic antibodies
3.1. Comparative quality studies
In the comparative quality study, quality attributes of a bio-
similar compared with those of its reference product are examined
extensively by the state-of-the-art analytical methods. The quality
66 A. Ishii-Watabe, T. Kuwabara / Drug Metabolism and Pharmacokinetics 34 (2019) 64e70

Risk Risk
Management plan Management plan

Compara ve clinical studies

Clinical studies • PK/PD
• PK/PD • Safety and efficacy
• Safety and efficacy • Immunogenicity
• Immunogenicity
Compara ve
non-clinical studies

Non-clinical studies Compara ve

quality studies
Quality Quality
characteriza ons characteriza ons
Innovator product Biosimilar
Fig. 1. Comparison of data requirements for approval of an innovator product and biosimilar. Typical strategy of innovator and biosimilar products was compared focusing on the
balance between studies in each product. The figure was prepared by modifying that from "Biosimilars in the EU" [33].

attributes to be compared include primary structure (i.e. amino
acid sequence and disulfide bond), higher order structure, post-
translational modification such as glycosylation site and glycan
structure, other modifications (e.g., oxidation, deamidation, and
glycation), charge isomers, product-related impurities (aggregates,
truncated forms, mismatched disulfide bond), and biological ac-
tivities. In cases of mAbs, biological activities of antigen-binding,
Fcg receptor binding, FcRn binding, antibody-dependent cellular
cytotoxicity (ADCC), and complement-dependent cytotoxicity
(CDC) should be evaluated.
The antigen binding affinity and specificity of the biosimilar and
the reference product should be comparable. However, the impact
of difference in biological activities related to Fc-regions on efficacy
and safety depends on the mode of action of the mAb. If the bio-
similar mAb exerts ADCC activities for its efficacy, the difference in
FcgRIIIa binding and ADCC activity need to be carefully assessed. In
case ADCC activity is not included in the mode of action of the mAb
(e.g., mAb against soluble monomer antigen), the difference in
FcgRIIIa binding may possibly not have a significant impact on the
efficacy and safety of the biosimilar mAb.
As an example, comparability assessment of infliximab bio-
similar CT-P13 is described here. In the comparative quality studies,
some minor differences, compared with the reference product,
including FcgRIIIa binding, the level of fucosylation, and ADCC ac-
tivity, were recorded [9]. It is well known that non-fucosylated mAb
shows higher ADCC activities [10,11], and contents of non-
fucosylated glycan can be a critical quality attribute of mAbs.
However, the initial differences observed in ADCC were only seen
in vitro by using target cells overexpressing transmembrane TNFa
and by using enriched natural killer cells from Crohn's disease
patients. When ADCC activity was tested using whole blood or
isolated peripheral blood mononuclear cells, the difference in
fucosylation for CT-P13 and the reference product did not impact
ADCC. Based on the data of clinical study for rheumatoid arthritis, it
was thought that such difference did not bring clinically mean-
ingful differences [9].
Meanwhile, the exact downstream effects of anti-TNFa agents in
rheumatological diseases and in inflammatory bowel disease (IBD)
are potentially different, and the immunogenicity of the biosimilar
compared with that of the originator could probably be different in
different clinical settings [12]. Regarding the mechanism of action,
in rheumatoid arthritis, infliximab is thought to act predominantly
through the neutralization of soluble and transmembrane TNFa; in
other diseases, like Crohn's disease, signaling through trans-
membrane TNFa and Fcg R (triggering apoptosis or ADCC) may play
a more important role [13]. In such a case, extrapolation across
indications should carefully be examined. In the case of CT-P13
applied to Health Canada, IBD was not approved initially as an
indication, however, after submission of additional data including
post-marketing surveillance study for IBD, the indication was
finally approved. The post-marketing studies are also crucial to
provide useful insights into its efficacy and safety and to further
support the use of biosimilars.
Among the quality attributes of mAbs, potential PK-related
quality attributes are listed in Table 2. These include isoelectric
point, charge variant contents, glycan structure, antigen binding,
FcgR binding, and FcRn binding. Isoelectric point and charge
variant contents are related to non-specific cellular uptake of

Table 2
mAb quality attributes related to PK.

Quality attributes Typical in vitro evaluation methods Relationship with PK

Physicochemical property Isoelectric point Isoelectric focusing Cellular uptake

Biological property
(Contents of acidic and basic variants)
Met oxidation in CH2 domain LC/MS FcRn binging
High-mannose glycan Oligosaccharide analysis Mannose receptor binding (suggested)
Soluble antigen binding SPR, ELISA Disposition as immune complex
Cell-surface antigen binding SPR, ELISA, Flow cytometry Disposition via intracellular uptake
FcRn binding SPR Recycling
FcgR binding SPR Disposition via intracellular uptake or phagocytosis
 A. Ishii-Watabe, T. Kuwabara / Drug Metabolism and Pharmacokinetics 34 (2019) 64e70 67

mAbs [14]. Glycan structure attached in CH2 domain of IgG was patient population, such as cases with ankylosing spondylitis and
reported to affect the PK profile of mAbs. mAbs bearing high- rheumatoid arthritis. In the PLANETAS study [29], a Phase 1 ran-
mannose glycan shows shorter half-life [15,16]. Antigen binding domized, double-blind, multicenter, multinational, parallel-group
properties may affect the PK profile by accelerating the clearance study was conducted wherein patients with ankylosing spondy-
of immune complexes. Binding with cell surface antigen may litis were administered 5 mg/kg of CT-P13 or innovator infliximab
relate to the target-mediated drug disposition (TMDD) [17e20]. via 2 h intravenous infusion at 0, 2, 6, and then 8 weeks interval up
FcgR binding may accelerate the cellular uptake of antigen- to 30 weeks. Geometric means of Cmax and AUC at steady state
antibody complex [21,22]. Because higher ADCC is related to between weeks 22 and 30 were 147.0 mg/mL and 32765.8 mg h/mL
higher clearance of mAb, ADCC may enhance the disposition of for CT-P13 (n ¼ 113, 112), and 144.8 mg/mL and 31359.3 mg h/mL for
mAb [17,23]. Antibody-dependent cellular phagocytosis (ADCP) CT-P13 (n ¼ 110), respectively. As the ratio of geometric means was
may also be involved in the clearance of mAb. FcRn binding, which 104.5% for AUC and 101.5% for Cmax, 90% CI were within 0.94e1.16
leads to the recycling of mAbs, directly affects the PK profile of and 0.95 to 1.16.
mAbs [24,25]. Oxidation of methionine residues on CH2 domain In Japanese patients with rheumatoid arthritis [30], CT-P13 or
are known to reduce the FcRn binging [26,27], resulting in innovator infliximab were administrated via 2 h intravenous infusion
decrease in serum half-life. at 0, 2, 6, and then 8 weeks interval up to 54 weeks. Cmax and AUC
after the second administration (6e14 weeks) were evaluated and
90% CI were met with the bioequivalence criteria. These results
3.2. Pharmacokinetics of biosimilars in nonclinical studies
suggest that the differences in quality attributes between the bio-
similar and the reference product have little impact on their PK.
Similarities in PK parameters of biosimilars have been evaluated
These clinical trials exhibited the comparability of biosimilar product
in experimental animals such as mice, rats, and cynomolgus
with the reference product in PK, safety, and immunogenicity.
monkeys. As in the case of other biologics, monkeys are most
Following recent FDA guidance [31], which states that clinical PK and
frequently used for this assessment. As an example, PK parameters
PD of studies should be conducted in healthy subjects if the products
of infliximab biosimilar is shown in Table 3 [28]. Similarities of
can be safely administered to them, the PK, safety, immunogenicity
clearance and half-life of infliximab biosimilar to EU-product (i.e.,
of the successful biosimilar products were evaluated in healthy
innovator infliximab approved in Europe) were observed in mice,
subjects. The detailed PK parameters were calculated for biosimilars
rats, and cynomolgus monkeys, even in small numbers of animals.
and their reference product after their single administration. As ex-
Comparable PK parameters in animals were also confirmed for
amples, PK parameters of several biosimilar products, SB2 for
other approved biosimilars. Judging from the approved biosimilars’
infliximab [32], SB5 for adalimumab [33], PF-05280014 for trastu-
data, the comparability in biosimilar PK in human may be pre-
zumab [34], and PF-06439535 for bevacizumab [35] are shown in
dictable using animal data.
Table 4. These 4 biosimilars and the respective reference products
showed the typical PK features, such as low systemic clearance
3.3. Pharmacokinetics of biosimilars in clinical studies (10e20 mL/h), small distribution volumes (50e70 mL/kg), and long
half-lives (200e400 h). All PK parameters of the biosimilars were
PKs of the approved biosimilars of therapeutic antibodies are comparable to their reference products (US- and EU-product). In
summarized (Tables 4 and 5). Evaluating bioequivalence of bio- addition, the bioequivalence of these biosimilars were also met with
similars to the reference products was one of the required regula- the criteria, as for primary PK parameters, Cmax and AUC, 90% CIs of
tory practices. The PKs of biosimilars were compared with those of the geometric mean of the biosimilar to the reference product were
their reference (innovator) products, i.e., licensed and marketed completely within 0.8e1.25.
products in EU and United states (EU-product and US-product). Due Among the mentioned antibodies, infliximab and adalimumab
to the long-half lives and possible immunogenicity of therapeutic showed relatively high incidence in Anti-Drug Antibody (ADA)
antibodies, the clinical studies were set as parallel group design and production [32,33]. PK parameters of biosimilar product (SB5) or
not as a cross-over design. Generally, acceptance criterion for the innovator infliximab compared between ADA-positive group and
bioequivalence of primary PK parameters (peak concentration ADA-negative group are shown in Table 5. Either in ADA-positive
(Cmax) and area under the concentration-time curve (AUC)) was group or ADA-negative group, PK parameters of biosimilar were
that the 90% confidence intervals (CIs) for the ratio of the geometric comparable to the reference products. However, obvious differ-
mean of the biosimilar to the reference product should be ences were observed in PK parameters between ADA-positive and
completely within 0.8e1.25. negative group. The half-life of biosimilar was 218 h in ADA-
As to the first antibody biosimilar product of infliximab (CT- positive group, which was almost half of that in ADA-negative
P13), PK, efficacy, and safety were simultaneously evaluated in the group (411 h). The clearance (CL) and distribution volume (Vd) in
ADA-positive group were 1.3-fold and 0.7-fold of ADA-negative
group, respectively, than those in ADA-negative group. The same
Table 3
Pharmacokinetic parameters in male experimental animals after single intravenous
alteration in PK parameters was observed in both the reference
administration of 10 mg/kg of infliximab biosimilar or EU-product. compounds (US- and EU-product). Similar results were observed in
adalimumab biosimilar and its reference compound [36]. For tras-
Animal AUC CL t1/2b
tuzumab and bevacizumab, there was no data to compare PK pro-
mg$h/mL mL/h/kg h files in ADA-positive groups since the incidence of ADA production
biosimilar mouse (Tg197) 16.3 ± 4.9 0.61 ± 0.2 113 ± 49 was rare. These results indicate that the ADA production signifi-
EU-product 13.6 ± 6.8 0.74 ± 0.3 135 ± 61 cantly affects PK of the antibodies, and results in higher clearance
biosimilar mouse (B6CBA) 19.0 ± 5.4 0.53 ± 0.3 168 ± 29
and shorter half-life. In the case of infliximab and adalimumab,
EU-product 24.3 ± 7.3 0.40 ± 0.1 216 ± 57
biosimilar rat 51.0 ± 3.5 0.20 ± 0.01 296 ± 63 there were no differences in the incidence of ADA production be-
EU-product 52.1 ± 2.6 0.19 ± 0.01 335 ± 34 tween biosimilar and reference product. Thus, ADA production did
biosimilar cynomolgus monkey 29.0 ± 4.4 0.35 ± 0.05 150 ± 54 not affect the comparability of these biosimilars. However, in
EU-product 29.5 ± 10.1 0.36 ± 0.10 153 ± 48 general, if a biosimilar exhibits new immunogenicity compared
Each value represents mean ± S.D. (n ¼ 6). with the reference, the comparability could not be assured. As a
68 A. Ishii-Watabe, T. Kuwabara / Drug Metabolism and Pharmacokinetics 34 (2019) 64e70

Table 4
Pharmacokinetic parameters in human healthy volunteers after single administration of biosimilar product or its original antibody products.

Product n Tmax Cmax AUC0e∞ CL Vd t1/2

h mg/mL mg$h/mL mL/h or mL/h/kg mL or mL/kg h

Infliximaba biosimilar 51 3.0 (2.0e6.0) 127.0 ± 16.9 38703 ± 11114 10.9 ± 3.2 mL/h 4587 ± 1583 mL 324.1 ± 148.7
EU-product 53 2.1 (2.0e6.1) 126.2 ± 17.9 39360 ± 12332 11.1 ± 3.0 4846 ± 1287 339.5 ± 155.4
US-product 53 3.0 (2.0e6.1) 129.2 ± 18.8 39270 ± 10064 10.7 ± 2.9 4806 ± 1216 339.7 ± 135.6
Adalimumabb biosimilar 62 144 (24e505) 3.37 ± 0.98 2406 ± 826 19.0 ± 7.8e mL/h 8279 ± 2564f mL 342 ± 153
EU-product 61 144 (24e338) 3.55 ± 1.18 2436 ± 916 18.6 ± 6.5e 8558 ± 3311f 358 ± 181
US-product 57 144 (24e336) 3.49 ± 957 2423 ± 957 19.0 ± 7.3e 8639 ± 3658f 340 ± 134
Trasutuzumabc biosimilar 34 159 ± 26 37130 ± 6305 0.166 ± 0.026 mL/h/kg 56.1 ± 8.2 mL/kg 213 ± 42
EU-product 35 174 ± 31 40330 ± 6994 0.153 ± 0.025 51.7 ± 6.9 220 ± 42
US-product 32 164 ± 31 37310 ± 6728 0.166 ± 0.032 55.7 ± 8.8 212 ± 47
Bevacizumabd biosimilar 32 142.9 ± 20.3 43080 ± 7103 0.119 ± 0.021 mL/h/kg 62.4 ± 10.6 mL/kg 397 ± 63
EU-product 33 137.0 ± 20.5 43830 ± 8326 0.117 ± 0.022 64.9 ± 9.6 417 ± 90
US-product 32 130.0 ± 18.2 41450 ± 5350 0.122 ± 0.016 67.7 ± 7.7 413 ± 57
a
Antibody (5 mg/kg) was intravenously infused for 2 h.
b
Antibody (40 mg) was subcutaneously administered.
c
Antibody (6 mg/kg) was intravenously infused for 90 min.
d
Antibody (5 mg/kg) was intravenously infused for 90 min.
e
CL/F.
f
Vd/F.

Table 5
Impact of ADA production on the pharmacokinetic parameters of infiliximab in human healthy volunteers after 5 mg/kg single intravenous infusion for 2 h.

ADA n Tmax Cmax AUC0e∞ CL Vd t1/2

h mg/mL mg$h/mL mL/h mL h

biosimilar positive 23 3.0 (2.0e6.0) 123.9 ± 14.0 31523 ± 7376 12.7 ± 3.4 3643 ± 1473 218.3 ± 111.1
negative 28 3.0 (2.1e6.0) 129.6 ± 18.8 44601 ± 10218 9.4 ± 2.0 5362 ± 1222 411.0 ± 116.3
EU product positive 20 3.0 (2.0e6.1) 124.6 ± 14.5 30808 ± 5468 13.6 ± 2.0 3915 ± 1210 205.5 ± 77.2
negative 33 2.1 (2.0e6.0) 127.2 ± 19.8 44543 ± 12489 9.5 ± 2.5 5411 ± 975 420.6 ± 133.1
US product positive 20 3.0 (2.0e6.1) 128.2 ± 17.6 31991 ± 7142 12.9 ± 2.8 4110 ± 1263 236.8 ± 101.5
negative 33 2.1 (2.0e6.0) 129.8 ± 19.7 43682 ± 9006 9.4 ± 2.0 5228 ± 984 402.1 ± 114.7

Each value represents mean ± S.D.

predictive method of immunogenicity in human, in vitro compar-
ative immunogenicity assessment (IVCIA) assay has been devel-
oped [37]. Such method can be used for the comparative
assessment of immunogenicity of mAbs with different amino acid
sequence in early phase of drug development, however, it is less
useful for comparing the immunogenicity of innovator and bio-
similar mAbs, which relates to the quality of these products. At
present, there is no reliable predictive methodology to evaluate the
immunogenicity of biologics in humans, making it very important
to develop such methodology for efficient development of
biosimilars.
3.4. Considerations in bioanalytical aspects
In the comparability assessment of PK profile, validity of bio-
analytical method used for drug concentration analysis is critical.
Typically, a ligand-binding assay such as ELISA is used. The basic
requirements for the bioanalytical methods in biosimilar devel-
opment is same as that described in bioanalytical method vali-
dation guideline [38e40]. The items required in validation are
specificity, selectivity, calibration curve, accuracy, precision,
dilution linearity, and stability. The analytical performance should
meet the criteria described in the guideline. Critical reagents
including binding protein directly to the analyte should be qual-
ified appropriately.
The important issue in bioanalysis using ligand-binding assay
in comparative non-clinical and clinical studies is reactivity of
binding reagents to the analytes (i.e. reference product and
biosimilar). Macromolecules such as mAbs are heterogeneous
and their immunoreactivity may vary depending on the
manufacturing process. It is important that binding properties of
the binding reagent used in comparative PK studies with a bio-
similar and the reference product is equivalent, i.e. dose
response curve for both biosimilar and its reference product is
identical.
Bioanalysis of biosimilar evaluation can be performed via a one
assay or two assays approach [41] (Fig. 2). If results from the cali-
bration curve similarity, accuracy, and precision testing performed
above are satisfactory, with no outstanding concerns identified, a
single assay with one calibration curve can be used. However, if the
results from that testing are not satisfactory or a concern is iden-
tified, then the two drugs may not be considered bioanalytically
similar and must be further investigated. Upon investigation, if no
tangible cause for the difference in the performance of the two
drugs is identified, two separate assays may be used (with appro-
priate justification) in the interest of progressing the drug devel-
opment program [41]. This is because quality attributes considering
the efficacy and safety are different from binding properties to
binding reagent used for bioanlaysis. Such cases are often noted for
glycoprotein with complex glycan structure, and mAbs are less
likely to show such differences.
The most optimal approach is to develop a single PK assay, using
a single analytical standard, for quantitative measurement of the
biosimilar and reference products in serum matrix. Use of a single
PK assay for quantification of multiple products requires a scien-
tifically sound testing strategy to evaluate bioanalytical compara-
bility of the test products within the method, and provide a solid
data package to support the conclusions [42].
The same consideration is necessary when ADA is evaluated
[43]. In the case of immunogenicity assessment, cut point
 A. Ishii-Watabe, T. Kuwabara / Drug Metabolism and Pharmacokinetics 34 (2019) 64e70 69

One assay Two assays

An gen used for
binding reagent Biosimilar Reference product Biosimilar
prepara on
Reference
Biosimilar Reference product Biosimilar
standard
Reference
Biosimilar Reference product Biosimilar
product

Study samples
in biological
matrices

Fig. 2. Bioanalytical platforms for comparative PK studies. One assay composed of one set of binding reagents prepared by using one (typically biosimilar) product, and same set of
binding reagents are used for both innovator and reference products. Two assays means two sets of assays. One is for innovator and the other is for biosimilar.

setting is critical, therefore, it is quite difficult to keep the
analytical performance of two assays the same. As is the case for
PK assay, ADA assay should also aim to detect all ADA against
biosimilars.
4. Concluding remarks/future perspective
Development of biosimilar mAbs is expected to decrease the
medical expenses and make it easier for patients to access medi-
cines needed for treatment. In 2017, EMA released the information
guide for health care professionals [44] and for patients [45]. These
documents provide reference information on both the science and
regulation of the use of biosimilars. In 2018, FDA released Biosimilar
Action Plan encouraging innovation and competition among bi-
ologics and the development of biosimilars [46]. From a scientific
viewpoint, elucidating the factors regulating the PK profile of mAbs
and other biologics in humans may contribute to more efficient
development of biosimilars and next generation innovative mAbs.
These factors include both product quality attributes and patient
features. As future perspective for biosimilars, studies on critical
quality attributes and critical clinical attributes related to safety and
efficacy of the products are important, which will also be necessary
for efficient development of innovative mAbs.
Authorship contributions
Wrote or contributed to the writing of the manuscript: AI and
TK.
Conflicts of interest
None.
Acknowledgement
This study was supported in part by AMED (Japan Agency for
Medical Research and Development) under Grant Number
JP17mk0101039 and JP18mk0101104.
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