International Journal of

Molecular Sciences

Communication
Exploring the Combined Action of Adding Pertuzumab to
Branded Trastuzumab versus Trastuzumab Biosimilars for
Treating HER2+ Breast Cancer
Emma Franco-Mateos 1 , Virginia Souza-Egipsy 1 , Laura García-Estévez 2 , José Pérez-García 3,4,5 , María Gion 6 ,
Laia Garrigós 3 , Patricia Cortez 7 , Cristina Saavedra 6 , Patricia Gómez 3 , Carolina Ortiz 3 , Víctor L. Cruz 1 ,
Javier Ramos 1 , Javier Cortés 3,4,8,† and Juan F. Vega 1, *

1 BIOPHYM, Department of Macromolecular Physics, Instituto de Estructura de la Materia, IEM-CSIC,

C/Serrano 113 bis, 28006 Madrid, Spain; [email protected] (E.F.-M.);
[email protected] (V.S.-E.); [email protected] (V.L.C.); [email protected] (J.R.)
2 Breast Cancer Department, MD Anderson Cancer Center, 28033 Madrid, Spain; [email protected]
3 International Breast Cancer Center (IBCC), Pangaea Oncology, Quiron Hospital, 08017 Barcelona, Spain;
[email protected] (J.P.-G.); [email protected] (L.G.); [email protected] (P.G.);
[email protected] (C.O.); [email protected] (J.C.)
4 Medica Scientia Innovation Research (MedSIR), 08018 Barcelona, Spain
5 Medica Scientia Innovation Research (MedSIR), Ridgewood, NJ 07450, USA
6 Medical Oncology Department, Ramón y Cajal University Hospital, 28034 Madrid, Spain;
[email protected] (M.G.); [email protected] (C.S.)
7 IOB, Institute of Oncology, 28007 Madrid, Spain; [email protected]
8 Faculty of Biomedical and Health Sciences, Department of Medicine, Universidad Europea de Madrid,
28670 Madrid, Spain
* Correspondence: [email protected]
† J.C. was at Vall d’Hebron Institute of Oncology, 08035 Barcelona, Spain, during the development of this work.

Citation: Franco-Mateos, E.; Abstract: The binding activity of various trastuzumab biosimilars versus the branded trastuzumab
Souza-Egipsy, V.; García-Estévez, L.; towards the glycosylated extracellular domain of the human epidermal growth factor receptor 2
Pérez-García, J.; Gion, M.; Garrigós, L.; (HER2) target in the presence of pertuzumab was investigated. We employed size exclusion chro-
Cortez, P.; Saavedra, C.; Gómez, P.; matography with tetra-detection methodology to simultaneously determine absolute molecular
Ortiz, C.; et al. Exploring the
weight, concentration, molecular size, and intrinsic viscosity. All trastuzumab molecules in solution
Combined Action of Adding
exhibit analogous behavior in their binary action towards HER2 regardless of the order of addition of
Pertuzumab to Branded Trastuzumab
trastuzumab/pertuzumab. This analogous behavior of all trastuzumab molecules, including biosimi-
versus Trastuzumab Biosimilars for
lars, highlights the robustness and consistency of their binding activity towards HER2. Furthermore,
Treating HER2+ Breast Cancer. Int. J.
Mol. Sci. 2024, 25, 3940. https://
the addition of HER2 to a mixture of trastuzumab and pertuzumab leads to increased formation
doi.org/10.3390/ijms25073940 of high-order HER2 complexes, up to concentrations of one order of magnitude higher than in the
case of sequential addition. The observed increase suggests a potential synergistic effect between
Academic Editors: Menotti Ruvo and
these antibodies, which could enhance their therapeutic efficacy in HER2-positive cancers. These
Annamaria Sandomenico
findings underscore the importance of understanding the complex interplay between therapeutic an-
Received: 22 February 2024 tibodies and their target antigens, providing valuable insights for the development of more effective
Revised: 18 March 2024 treatment strategies.
Accepted: 29 March 2024
Published: 1 April 2024 Keywords: HER2+ breast cancer; monoclonal antibodies; trastuzumab biosimilars

Copyright: © 2024 by the authors.

1. Introduction
Licensee MDPI, Basel, Switzerland.
This article is an open access article Monoclonal antibodies (mAbs) have revolutionized cancer treatment over recent years,
distributed under the terms and offering precise targeting to specific proteins or cells. In the case of breast cancer, several
conditions of the Creative Commons mAbs-based therapies have been developed and approved for use [1]. Notable among
Attribution (CC BY) license (https:// these are trastuzumab and pertuzumab, and more recently tucatinib and trastuzumab
creativecommons.org/licenses/by/ deruxtecan [2]. These drugs target the very aggressive human epidermal growth factor
4.0/). receptor 2-positive (HER2+) cancer cells by blocking the action of the HER2 protein [3,4].

Int. J. Mol. Sci. 2024, 25, 3940. https://doi.org/10.3390/ijms25073940 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2024, 25, 3940 2 of 14

The HER2 transmembrane protein is a member of the family of epithelial growth factor
receptors, the overexpression of which is related with aggressive carcinomas. Presently,
the 5-year survival rate is increasing up to 90% in HER2+ early breast cancer treated with
chemotherapy and combined or dual mAbs trastuzumab and pertuzumab therapies, as
they show complementary mechanisms of action [5].
Clinical trials and meta-analyses have reported cases of sequential addition in patients
undergoing dual anti-HER2 therapies. Jagosky and Liu [6,7] have recently reviewed several
studies that support the substantial improvement in the outcome of HER2+ breast cancer
patients with combined therapy of pertuzumab and trastuzumab [8–17].
Additionally, it has been demonstrated that the combination of trastuzumab emtansine
(T-DM1) and pertuzumab exhibits synergistic cytotoxic activity in cell culture and enhanced
antitumor activity [18]. Moreover, the results indicated that the safety of dual-target therapy
is similar to that of single-target therapy [11,19].
On the other hand, biosimilars highly resemble the branded drugs, but with the
advantage of quicker development, lower cost of production, and greater availability. As the
development process of a biosimilar is somewhat different to that of the reference product,
it is possible to include minor changes or additions to the protein structure that can produce
a drug with different efficacy or side effect profiles. However, a biosimilar drug may not
be an exact copy of the reference product, but it must maintain identical or even improve
its mechanisms of action, dosage and route of administration, and effectiveness [20–22].
Several biosimilars of Herceptin® [Genentech, Inc., South San Francisco, CA, USA] have
been approved for use in breast cancer treatment, including Ontruzant® [N.V. Organon,
Jersey City, NJ, USA] and Herzuma® [Celltrion, Inc., Inchon, South Korea] [23–25].
In this study, we assessed the ability of each of these antibodies to bind to HER2 in
the presence of the pertuzumab Perjeta® [Genentech, Inc.], and investigated the order of
addition of the mAbs to HER2 in solution. The selected methodology for investigating
the binding process was size exclusion chromatography with tetra-detection (SEC-TD)
in solution. We acknowledge that alternative techniques, such as nuclear magnetic reso-
nance, isothermal titration calorimetry, and analytical ultracentrifugation, could potentially
yield complementary results. This analytical approach offers numerous advantages for
examining protein–protein interactions or the binding between mAbs and target proteins.
SEC-TD is fast, efficient, cost-effective, and sensitive, enabling the simultaneous detection
of proteins and complexes in a single analytical run. Additionally, it has been reported that
binding experiments in solution may yield more realistic results compared to protocols
using antibody support techniques [26]. This approach facilitates the measurement of
composition, absolute molecular weight and size, concentration, and molecular density or
intrinsic viscosity of biomacromolecules, providing a comprehensive overview of protein-
protein interactions and the stability of formed complexes [27–31]. To the best of our
knowledge, such vital information is lacking in the scientific literature, thereby constituting
an essential test for assessing the biophysical comparison of biosimilar antibodies. Our
findings could provide insights into potential combination therapies for HER2+ breast
cancer and contribute to the optimization of treatment efficacy.

2. Results
The hydrodynamic and electrostatic characterization results of the mAbs are presented
in Figures S1–S3 and Tables S1 and S2 of the Supplementary Material archive. The findings
depicted in Figures S1–S3 indicate a considerable level of resemblance among the examined
mAbs. Specifically, the diffusion coefficient (representing hydrodynamic size) exhibits
variances of no more than 2% across the samples, indicating a closely matched, if not
indistinguishable, morphology or shape of the mAbs. The reported positive value of
the net charge, Z, for the mAbs at pH 7.5 is in the same range as those values reported
in other studies for IgG1 antibody at pH within 5.0 and 9.0 and low ionic strength. A
difference is found between trastuzumab and pertuzumab, explained by the differences in
their amino acid sequences (AAS). No appreciable differences have been found between
 a closely matched, if not indistinguishable, morphology or shape of the mAbs. The
reported positive value of the net charge, Z, for the mAbs at pH 7.5 is in the same range
as those values reported in other studies for IgG1 antibody at pH within 5.0 and 9.0 and
low ionic strength. A difference is found between trastuzumab and pertuzumab,
Int. J. Mol. Sci. 2024, 25, 3940 explained by the differences in their amino acid sequences (AAS). No appreciable 3 of 14
differences have been found between trastuzumab and the two biosimilars, Herzuma and
Ontruzant, in this aspect. The thermal stability results, as depicted in Figures S4 and S5,
trastuzumab
yielded valuableand the two biosimilars,
insights Herzuma
into the capacity of theand Ontruzant,
mAbs to upholdin their
this aspect. The integrity
structural thermal
stability results, as depicted
and functionality in Figures of
across a spectrum S4temperatures.
and S5, yieldedNotably,
valuablenoinsights into the
discernible capacity
disparities
of
in the mAbswere
stability to uphold theiramong
observed structural
the integrity and functionality
various antibodies tested, across a spectrum
indicating consistent of
temperatures. Notably, no discernible
resilience across the antibody samples. disparities in stability were observed among the
various antibodies
Herceptin andtested, indicating consistent
the biosimilars resilience
of trastuzumab, acrossOntruzant
namely, the antibody samples.
and Herzuma,
Herceptin and the biosimilars of trastuzumab, namely, Ontruzant and
exhibit identical SEC-TD profiles, as demonstrated in Figure 1. Table 1 outlines the main Herzuma, ex-
hibit identical
molecular andSEC-TD profiles, as demonstrated
physical–chemical characteristicsinofFigure 1. Table
the mAbs 1 outlines
and the main
the glycosylated
molecular
extracellular domain of HER2 (g-eHER2) utilized in this study. These propertiesextra-
and physical–chemical characteristics of the mAbs and the glycosylated were
cellular
acquired domain of HER2
from the SEC-TD(g-eHER2)
outcomes utilized in this
depicted study. These
in Figure 1 and properties weresca
dynamic light acquired
ering
from
(DLS)the SEC-TD outcomes
experiments, conducteddepicted in Figure 1 of
at a temperature andT =dynamic
309 K (36light
°C)scattering (DLS)
as described ex-
in the
periments, conducted at a temperature of T = 309 K (36 ◦ C) as described in the Methods
Methods section (see also Supplementary Material, in which the complete
section (see also Supplementary
characterization Material, in which the complete characterization of the
of the mAbs is described).
mAbs is described).

Figure 1. SEC
Figure 1. SEC traces
traces for
for the
the trastuzumab
trastuzumab Herceptin
Herceptin and
and the
the biosimilars
biosimilars Ontruzant
Ontruzant and
and Herzuma.
Herzuma.
Four
Four detectors—refractive index: RI; ultraviolet: UV; right-angle light sca ering: RALS; and
detectors—refractive index: RI; ultraviolet: UV; right-angle light scattering: RALS; and low-
low-
angle · −1
angle light sca ering: LALS—of the biosimilars studied for a concentration of 1.0 mg·mL−1 at
light scattering: LALS—of the biosimilars studied for a concentration of 1.0 mg mL at aa
temperature
temperatureof ofTT== 309
309 K
K are
are indicated.
indicated.

Table1.1.Molecular
Table Molecular and
and hydrodynamic
hydrodynamic properties
properties of
of the
themAbs mAbsand
andtarget
targetHER2;
HER2;molecular
molecularweight
weight
(Mww), intrinsic
(M intrinsic viscosity
viscosity ([η]),
([η]), hydrodynamic
hydrodynamicradius
radius(r(rhh),), and
and UV
UV coefficient
coefficient ofof absorption
absorption at
at 280
280 nm
nm
(dA/dc).
(dA/dc).

[η]
[η] 10 2 10 2
rrhh dA/dc
dA/dc
Mw Mw 3 − −1 ·mL·cm−1
−1 )
Sample
Sample ·g 13·g
(cm (cm ) −1) (nm)
(nm) (g ·mL·cm )
(g−1
(kDa)(kDa)
± 0.2± 0.2
s.d. s.d. s.d. ±
s.d. 0.1
± 0.1 s.d. ±±0.02
s.d. 0.02
Perjeta
Perjeta ± 0.8 ± 0.8
147.1 147.1 6.4 6.4 5.5
5.5 1.33
1.33
Herceptin
Herceptin 147.0 147.0
± 1.6 ± 1.6 6.5 6.5 5.5
5.5 1.38
1.38
Herzuma
Herzuma ± 1.4 ± 1.4
147.9 147.9 6.3 6.3 5.5
5.5 1.38
1.38
Ontruzant
Ontruzant ± 1.3 ± 1.3
147.7 147.7 6.4 6.4 5.5
5.5 1.37
1.37
g-eHER2
g-eHER2 86.3 ±86.3
1.0 ± 1.0 6.5 6.5 4.4
4.4 0.90
0.90
Standard deviation values have been obtained from 5 to 10 independent measurements under the same conditions.
Theoretical molecular weight values for the mAbs are 145.4 and 145.2 kDa for trastuzumab and pertuzumab,
respectively. The experimental value obtained for g-eHER2 includes glycosylation.

In this study, we investigated the binding activity of trastuzumab biosimilars with

the HER2 receptor in both binary and ternary interactions. Firstly, we examined the inter-
actions involving either HER2 with trastuzumab or HER2 with pertuzumab individually.
Subsequently, we explored the synergistic effects of combining both trastuzumab and per-
Int. J. Mol. Sci. 2024, 25, 3940 4 of 14

tuzumab antibodies with the HER2 receptor. To analyze these interactions, we introduced
HER2 to an excess of monoclonal antibodies (mAbs) in three different scenarios: (1) adding
HER2 to trastuzumab followed by pertuzumab, (2) adding HER2 to pertuzumab followed
by trastuzumab, and (3) adding HER2 to a 1:1 mixture of both mAbs. Cases (1) and (2)
involved a two-step process, first the binary interaction followed by the ternary interaction,
while case (3) focused solely on the ternary interaction. In all cases, we maintained a
consistent mAbs/HER2 ratio of 3:1. Molecular features of the resulting complexes from
each step, including binary (C1 and C2 complexes) and ternary (C3 complex) interactions,
were measured and presented in Tables 2–4.

Table 2. Molecular and hydrodynamic properties of the complexes (case 1); molecular weight,
intrinsic viscosity, hydrodynamic radius, and UV coefficient of absorption at 280 nm.

[η] 102 rh dA/dc

Sample Mw
(cm3 ·g−1 ) (nm) (g−1 ·mL·cm−1 )
C1/C2 (kDa)
s.d. ± 0.2 s.d. ± 0.1 s.d. ± 0.02
HRC/HER2 235.7/310.8 6.7/7.9 6.2/7.1 1.22/1.15
ONT/HER2 237.5/313.6 6.9/7.7 6.2/7.2 1.23/1.14
HZM/HER2 238.2/315.7 6.8/7.8 6.2/7.2 1.22/1.15
C3 Case 1: TZM/HER2/PZM
HRC/HER2/PJT 482.5 8.1 8.5 1.22
ONT/HER2/PJT 483.2 8.2 8.6 1.22
Standard deviations are obtained from the values of Mw by matching concentration in RI and UV detectors.
Theoretical Mw is around for C1, C2, and C3 are 234, 320, and 464 kDa, respectively, by assuming the values of
Mw in Table 1. HRC, ONT, HZM, and PJT stand for Herceptin, Ontruzant, Herzuma, and Perjeta, respectively.

Table 3. Molecular and hydrodynamic properties of the complexes (case 2); molecular weight,
intrinsic viscosity, hydrodynamic radius, and UV coefficient of absorption at 280 nm.

[η] 102 rh dA/dc

Sample Mw
(cm3 ·g−1 ) (nm) (g−1 ·mL·cm−1 )
C2 (kDa)
s.d. ± 0.2 s.d. ± 0.1 s.d. ± 0.02
PJT/HER2 310.0 8.0 7.4 1.12
C3 Case 2: PZM/HER2/TZM
PJT/HER2/HRC 484.6 8.1 8.5 1.22
PJT/HER2/ONT 481.8 8.2 8.5 1.23
PJT/HER2/HZM 491.6 8.1 8.6 1.22
Standard deviations are obtained from the values of Mw by matching concentration in RI and UV detectors.
Theoretical Mw is around for C1, C2, and C3 are 234, 320, and 464 kDa, respectively, by assuming the values of
Mw in Table 1.

Table 4. Molecular and hydrodynamic properties of the complexes (case 3); molecular weight,
intrinsic viscosity, hydrodynamic radius, and UV coefficient of absorption at 280 nm.

[η] 102 rh dA/dc

Mw
Sample (cm3 ·g−1 ) (nm) (g−1 ·mL·cm−1 )
(kDa)
s.d. ± 0.2 s.d. ± 0.1 s.d. ± 0.02
C3 Case 3: TZM/PZM/HER2
PJT-HRC/HER2 932.0/489.0 9.8/8.0 9.5/8.7 1.23/1.23
PJT-ONT/HER2 945.0/489.0 n.d./8.2 n.d./8.8 1.25/1.25
Standard deviations are obtained from the values of Mw by matching concentration in RI and UV detectors of the
corresponding peaks. Theoretical Mw is around for C1, C2, and C3 are 234, 320, and 464 kDa, respectively, by
assuming the values of Mw in Table 1.

It is important to note that the binding affinity of trastuzumab and pertuzumab in

their binary interaction to HER2 differs in solution (Figure 2). When HER2 is added to an
excess of trastuzumab, it mainly forms a heterodimer (C1, one HER2 domain bound to one
 Standard deviations are obtained from the values of Mw by matching concentration in RI and UV
detectors of the corresponding peaks. Theoretical Mw is around for C1, C2, and C3 are 234, 320,
and 464 kDa, respectively, by assuming the values of Mw in Table 1.

Int. J. Mol. Sci. 2024, 25, 3940 It is important to note that the binding affinity of trastuzumab and pertuzumab5 of in14
their binary interaction to HER2 differs in solution (Figure 2). When HER2 is added to an
excess of trastuzumab, it mainly forms a heterodimer (C1, one HER2 domain bound to
one
mAbmAb Fab,Fab, Figure
Figure 3A)3A) with
with a molecular
a molecular weightofofMM
weight w = 235.0–238.0 kDa. Additionally,
w = 235.0–238.0 kDa. Additionally, a
a HER2/mAb/HER2
HER2/mAb/HER2 heterotrimer
heterotrimer (C2, two HER2 boundstotothe
(C2, two HER2 bounds thetwo
twoavailable
availablemAb
mAbFabs,Fabs,
Figure 3B) is formed with M = 310–316 kDa. On the other hand, pertuzumab
Figure 3B) is formed with Mw = 310–316 kDa. On the other hand, pertuzumab is more
w is more
likely
likely to form
to form the heterotrimer
the heterotrimer (C2, Figure
(C2, Figure 3E) with
3E) with both both
Fabs Fabs
of theofmAb
the mAb
bound bound
to two toHER2
two
HER2
copies,copies, resulting
resulting in aofvalue
in a value Mw =of310.0
Mw ±= 310.0 ± 5.0
5.0 kDa kDa 2,(Figure
(Figure 2, right
right panel). panel).
The The
biosimilars
biosimilars
OntruzantOntruzant
and Herzumaand Herzuma showed identical
showed identical profiles profiles to Herceptin
to Herceptin in the in the binding
binding binary
binary stepHER2
step with with extracellular
HER2 extracellular
domain,domain,
as they as
alsothey also mostly
formed formedthe mostly
same the same
complexes
complexes in nearly identical proportions, as shown in Figure 2 (left panel). We
in nearly identical proportions, as shown in Figure 2 (left panel). We measured the sizes measured
the
of sizes
the two of complexes
the two complexes
(C1 and C2)(C1 to
and
be C2) to be around
different, different, 6.2around
and 7.36.2nm,and 7.3 nm,
respectively
respectively
(see Tables 2(seeandTables
3). 2 and 3).

Figure 2. SEC traces of mAb/HER2 complexes in binary interaction. SEC traces (RI signal) of com-
Figure 2. SEC traces of mAb/HER2 complexes in binary interaction. SEC traces (RI signal) of
Int. J. Mol. Sci. 2024, 25, x FOR PEER plexes
REVIEW 6 of 14
between each trastuzumab biosimilar and HER2 (left), and comparison of trastuzumab/HER2
complexes between each trastuzumab biosimilar and HER2 (left), and comparison of
and pertuzumab/HER2
trastuzumab/HER2 traces obtained in traces
and pertuzumab/HER2 the same conditions
obtained in the(right).
same conditions (right).

Figure 3. Schematic representation of the different complexes: HER2 extracellular domains (blue),
Figure 3. Schematic representation of the different complexes: HER2 extracellular domains (blue),
trastuzumab (T, black) and pertuzumab (P, grey). The different HER2 domains are indicated in
trastuzumab (T, black) and pertuzumab (P, grey). The different HER2 domains are indicated in (A).
(A).
(A,D)(A,D) represents
represents the complex
the complex 1 formed
1 formed by trastuzumab
by trastuzumab and pertuzumab
and pertuzumab with HER2,
with HER2, respec-
respectively;
tively; (B,E) represent the complex 2 for tratuzumab and pertuzumab with HER2, respectively;
(B,E) represent the complex 2 for tratuzumab and pertuzumab with HER2, respectively; and (C,F) and
(C,F) represent
represent the Complex
the Complex 3, in which
3, in which both both
mAbsmAbs are involved.
are involved.

We acknowledge that there are slight differences in the reported values of Mw for
different samples of complexes C1 and C2 in Table 2. While the range for each complex is
indeed relatively narrow (between 235.7 and 238.2 kDa for C1 and between 310.8 and 315.7
kDa for C2), there are variations that fall within the margin of accepted experimental
variability (less than 2%). This small variability in Mw does not significantly impact the
 Int. J. Mol. Sci. 2024, 25, 3940 6 of 14

We acknowledge that there are slight differences in the reported values of Mw for
different samples of complexes C1 and C2 in Table 2. While the range for each complex
is indeed relatively narrow (between 235.7 and 238.2 kDa for C1 and between 310.8 and
315.7 kDa for C2), there are variations that fall within the margin of accepted experimental
variability (less than 2%). This small variability in Mw does not significantly impact
the hydrodynamic radius values reported in our study. This observation is particularly
pertinent due to the compact conformation of the proteins, which minimizes changes in
hydrodynamic properties despite minor fluctuations in molecular weight. Furthermore, we
note that the hydrodynamic radius values for both C1 and C2 complexes are consistent with
the values of the molecular weight, with reported sizes of 6.2 nm and 7.2 nm, respectively.
In the next step of our study, by adding a second antibody to the binary solution for
the ternary interaction, larger complexes in all three cases are obtained (around a retention
volume of 13.5 mL). The Mw of these complexes is around 480–490 kDa, and there are
no differences found in the case of the biosimilars at this point. Figure 4 exemplifies this
result, showing case 2 of addition using pertuzumab (Perjeta) plus trastuzumab, Herceptin,
and the biosimilars. The main complex found at 13.5 mL has a molecular weight that is
consistent with the association of two antibodies (trastuzumab and pertuzumab) linked
through an HER2 copy, with the other Fab of pertuzumab also linked to a HER2 protein 7 of 14
Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW
unit (C3, see Figure 3F). Note that pertuzumab and trastuzumab bind to different HER2
domains (domain II and IV, respectively).

Figure 4. Comparison
Figure 4. Comparison ofof the
the SEC
SEC traces
tracesobtained
obtainedforforthe
thecomplexes
complexes inin case
case 2 for
2 for ternary
ternary interaction
inter-
Upper panel: panel:
action. Upper complete SEC-IR
complete traces
SEC-IR tracesofof the complexesand
the complexes and
freefree
mAbmAb for2: case
for case black 2: black
(HRC/HER2/PJT),
(HRC/HER2/PJT), red red(ONT/HER2/PJT),
(ONT/HER2/PJT), andand
blueblue (HZM/HER2/PJT).
(HZM/HER2/PJT). CurvesCurves have
have been been vertically
vertically
shifted forbetter
shifted for be ervisualization.
visualization.Bottom
Bo om panel:
panel: measured
measured absolute
absolute molecular
molecular weight
weight obtained
obtained for for the
complexes.
the complexes.

The
The production
productionofofthe theC3
C3complex
complexwithwitha amolecular
molecular weight of of
weight Mw M~480–490 kDa
w  480–490 kDa was
was examined in case 1, case 2, and case 3 experiments. The concentration of the C3
examined in case 1, case 2, and case 3 experiments. The concentration of the C3
heterocomplex over the total concentration of HER2 added was determined based on RI
heterocomplex overit the
detector signals, and wastotal
foundconcentration of HER2
that the differences in theadded was determined
production based on RI
of the C3 complex
detector
for case 2signals, and (Figure
experiments it was found
4) withthat
the the differences
different in thebiosimilars
trastuzumab production of the
were C3 complex
smaller
for case 2 experiments (Figure 4) with the different trastuzumab biosimilars were smaller
than 1%. The same trend was observed in case 1. Interestingly, in both cases 1 and 2, a
very small percentage (around 1–3%) of a very high molecular weight complex (Mw  930
kDa) was also detected around a retention volume of 12 mL.
In case 3 experiments, for which HER2 was added to a 1:1 mixture of trastuzumab
 Int. J. Mol. Sci. 2024, 25, 3940 7 of 14

than 1%. The same trend was observed in case 1. Interestingly, in both cases 1 and 2, a very
small percentage (around 1–3%) of a very high molecular weight complex (Mw ~930 kDa)
was also detected around a retention volume of 12 mL.
In case 3 experiments, for which HER2 was added to a 1:1 mixture of trastuzumab
and pertuzumab in a direct ternary interaction, a significant increase in the concentration
of high-order complexes of Mw = 930–940 kDa was measured, appearing at a retention
volume of 12 mL. The increase in the production of these complexes, with respect to cases 1
and 2, was over one order of magnitude (30%). Figure 5 provides a visual comparison
of the results obtained in cases 1, 2, and 3. The signals have been deconvoluted in order
to extract the concentration of each species. It is evident that a higher amount of high
Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW
molecular weight complexes was formed in case 3, and even a small amount of very high 8 of 14
molecular weight complexes of around 1400 kDa was detected, around a retention volume
of 10–11 mL.

Figure 5.
Figure Comparison ofofthe
5. Comparison theSEC
SECtraces obtained
traces for the
obtained for complexes in case
the complexes in1,case
2, and 3 for
1, 2, andternary
3 for ternary
interaction. Upper panels: SEC-IR traces of the complexes formed in the different casesstudy.
interaction. Upper panels: SEC-IR traces of the complexes formed in the different cases under under study.
Bottom
Bo om panel:
panel:Measured
Measured absolute molecular
absolute weightweight
molecular obtained for the complexes.
obtained C3 complexC3
for the complexes. appears
complex ap-
around 12 mL (grey zone).
pears around 12 mL (grey zone).
3. Discussion
3. Discussion
Previous works by our group presented a complete experimental and computational
Previous
modelling works
study bymolecular
of the our group presented
and a complete
hydrodynamic detailsexperimental
of both HER2andandcomputational
mAbs,
which agree with the results summarized in Table 1 [32,33]. Based on the
modelling study of the molecular and hydrodynamic details of both HER2 and mAbs,results presented,
all proteins
which agreeinwith
this study exhibitsummarized
the results the expected molecular
in Table 1and hydrodynamic
[32,33]. Based onproperties
the results pre-
consistent with their AAS. In addition to the basic molecular and hydrodynamic
sented, all proteins in this study exhibit the expected molecular and hydrodynamic prop-characteri-
zation, we have determined the extinction coefficient, dA/dc, of the proteins studied using
erties consistent with their AAS. In addition to the basic molecular and hydrodynamic
the UV signal to match protein concentration with that measured from the RI detector.
characterization, we have determined the extinction coefficient, dA/dc, of the proteins
Trastuzumab biosimilars (1.38 ± 0.02 mL·g−1 ) display a slightly higher value of dA/dc
studied using the UV signal to match protein concentration with that measured from the
RI detector. Trastuzumab biosimilars (1.38 ± 0.02 mL·g−1) display a slightly higher value
of dA/dc compared to pertuzumab (1.33 ± 0.02 mL·g−1), as expected due to the higher num-
ber of aromatic residues in the former. Although both trastuzumab and pertuzumab show
a high degree of chemical similarity, there are subtle differences in the amounts of tyrosine
Int. J. Mol. Sci. 2024, 25, 3940 8 of 14

compared to pertuzumab (1.33 ± 0.02 mL·g−1 ), as expected due to the higher number of
aromatic residues in the former. Although both trastuzumab and pertuzumab show a high
degree of chemical similarity, there are subtle differences in the amounts of tyrosine (Tyr),
tryptophan (Trp), and phenylalanine (Phe) residues, which can affect the extinction, ε, or
absorption coefficient, dA/dc, as measured by UV spectroscopy. The extinction coefficient
at 280 nm is unique to each protein and depends on the number of aromatic residues
as well as the solvent, temperature, and pH. We used the Protein Calculator Resource
(http://protcalc.sourceforge.net/, accessed on 31 March 2024) to theoretically compute
dA/dc for the samples under study, utilizing the corresponding AAS. The absorption
coefficients in water were estimated in this case using the method of Gill and von Hippel
at 280 nm [34]. We tested this procedure using bovine serum albumin, ovoalbumin, and
conalbumin protein standards. The experimental values obtained for dA/dc in our SEC-TD
system were 0.67, 0.72, and 1.12 mL·g−1 ·cm−1 , respectively, which are quite similar to those
calculated in silico for these proteins (0.69, 0.73, and 1.11, from the AAS) and reported
elsewhere [35]. The values obtained from the calculations for trastuzumab and pertuzumab
equal 1.41 and 1.37 mL·g−1 at 280 nm, respectively, in close agreement with those measured
experimentally.
It is evident that Herceptin and the biosimilars of trastuzumab, namely, Ontruzant and
Herzuma, exhibit strikingly similar characteristics across various parameters, including
hydrodynamic size, net charge, thermal stability, and extinction coefficient. Notably, the
hydrodynamic and electrostatic characterization results indicate a high level of resemblance
among the examined mAbs, with minimal variances observed in diffusion coefficients and
net charges. Furthermore, the SEC-TD profiles and molecular properties acquired from
DLS experiments reveal consistent molecular and physical–chemical characteristics among
Herceptin and its biosimilars. The negligible disparities observed in stability and extinction
coefficients between Herceptin and the biosimilars underscore their equivalence in terms of
structural integrity and biochemical properties. Therefore, based on the evidence presented,
it is reasonable to conclude that Herceptin and its biosimilars behave nearly identically,
further supporting their interchangeability in clinical practice.
In terms of interactions with HER2, our previous study found that pertuzumab Perjeta
has a stronger binding affinity to HER2 than trastuzumab Herceptin [36]. We explained this
difference by analyzing the different interfacial contact (IC) descriptors that contribute to
the estimated free energy of binding (∆Gbind ) value using a QSAR model. Additionally, we
conducted experiments that revealed that the pertuzumab IgG antibody binds to two HER2
proteins, one per Fab fragment, while trastuzumab mainly forms a monovalent complex.
We interpreted this finding using a geometrical model that identified steric crowding in the
trastuzumab/HER2 heterotrimer compared to the pertuzumab/HER2 heterotrimer. Now,
the result observed in Figure 2 demonstrates that the biosimilars Ontruzant and Herzuma
behave exactly the same as Herceptin in their binary interaction, as the concentration and
molecular weight profiles are identically replicated, indicating their equivalence in terms
of molecular weight and binding affinity to HER2. Small differences in the values for the
molecular features (Mw and rh ) and the previous results obtained in our group can be
anticipated [33]. We attribute the small changes to the typical experimental variability in
the case of Mw and to the slightly different conditions used in the case of rh and [η]. The [η]
values are in fact slightly lower now, which is a consequence of the higher temperature used
in the current set of experiments (T = 309 K) in comparison to that used in Reference [33].
Figures 2 and 4 suggest that the biosimilars Ontruzant and Herzuma have a similar
capacity as Herceptin for both the binary (C1 and C2) and the ternary (C3) interaction with
HER2. The results indicate that they are biologically equivalent to the reference drug to
bind HER2 in presence of pertuzumab and in the conditions under study. Notwithstanding,
it is interesting to note that when HER2 is added to the previously mixed trastuzumab
and pertuzumab antibodies (case 3 in Figure 5), a higher fraction of high-order complexes
is formed. In this case, a bivalent trastuzumab should be involved as a linker to form
the complex (Figure 6). In that way, the formed complexes of antigens and monoclonal
 Int. J. Mol. Sci. 2024, 25, 3940 9 of 14

antibodies linked via bivalent binding constitute molecular chains. The formation of such
antigen–antibody molecular catenaries was already proposed by Hughes-Jones et al., for
the synergistic lysis of red blood cells [37]. The reason for this extremely high avidity of
both mAbs together to form these high-order complexes with HER2 is at this moment
unknown, but the results suggest an increased availability of trastuzumab Fabs in order to
Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 10 of 14
form these complexes, or the appearance of altered intermolecular interactions for binding
HER2 to the two mAbs. Further studies are needed to fully understand the mechanisms
underlying these observations.

P/T + HER2
Figure 6.
Figure High-order complexes
6. High-order complexes (M(Mw ~930 kDa)
w  930 between
kDa) the mAbs
between trastuzumab,
the mAbs pertuzumab,
trastuzumab, and
pertuzumab, and
HER2. Pertuzumab
HER2. Pertuzumab again
againfavors C2C2
favors complex
complexin trastuzumab/HER2
in trastuzumab/HER2 interaction acting acting
interaction as linkers.
as linkers.
HER2 extracellular
HER2 extracellular domains
domains (blue), trastuzumab
(blue), trastuzumab (T, black), and pertuzumab
(T, black), (P, grey).
and pertuzumab (P,The different
grey). The different
domains of HER2 protein are indicated in different colors as in Figure
domains of HER2 protein are indicated in different colors as in Figure 3. 3.

The results obtained in this work settle that the coformulation of mixtures of thera-
The results obtained in this work se le that the coformulation of mixtures of thera-
peutic mAbs targeting multiple epitopes represents an appealing strategy to increase their
peutic
efficacymAbs targeting
[38,39] multiple
and approach the epitopes represents
goal of mimicking thean appealing
natural strategy
polyclonal to increase
humoral im- their
efficacy [38,39] and approach the goal of mimicking the natural polyclonal
mune response [40]. Although there are few systematic studies on this approach, reports on humoral im-
mune responsefor[40].
mAb mixtures Althoughofthere
the treatment arediseases
various few systematic
are emergingstudies
[41,42].on At this approach,
this respect, it reports
on mAb mixtures
is important for thethe
to understand treatment
potential of various diseases
interactions are emerging
between mAbs [41,42].
and the effects At this re-
of vari-
ables such as pH, concentration, temperature, and ionic strength [43].
spect, it is important to understand the potential interactions between mAbs and In the specific case of the ef-
the pertuzumab/trastuzumab
fects of variables such as pH,mixtures, it shouldtemperature,
concentration, be noted that aand fixed-dose combination
ionic strength [43]. In the
of pertuzumab and trastuzumab for subcutaneous injection (PHESGO™, F. Hoffmann-La
specific case of the pertuzumab/trastuzumab mixtures, it should be noted that a fixed-
Roche Ltd., Basel, Switzerland) has recently been approved by the U.S. Food and Drug
dose combination of pertuzumab and trastuzumab for subcutaneous injection
Administration (FDA) and the European Medicines Agency. These findings have important
(PHESGO™,
implications for F. the
Hoffmann-La
developmentRoche of thisLtd.,
type Basel, Swi mAb
therapeutic erland) has recently
combinations [44].been approved
by the U.S.our
While Food
studyand Drug Administration
provides (FDA)
valuable insights into and the European
the similarities Medicines
between Herceptin andAgency.
These findingsashave
its biosimilars, wellimportant implications
as their interactions for theitdevelopment
with HER2, is important toof this type
recognize therapeutic
certain
mAb combinations
limitations. [44].
Firstly, our investigation focused primarily on physicochemical parameters
and While
bindingouraffinity,
studyleaving
providesout potential
valuable functional assays
insights into thethat could provide
similarities betweenfurther
Herceptin
understanding of therapeutic efficacy. Additionally, this study was
and its biosimilars, as well as their interactions with HER2, it is important to recognize conducted under
specificlimitations.
certain experimentalFirstly,
conditions,
our and variations in
investigation factors such
focused as pH,on
primarily temperature, and
physicochemical pa-
ionic strength could influence results. Moving forward, it is beneficial to explore these
rameters and binding affinity, leaving out potential functional assays that could provide
mAbs’ efficacy in preclinical models or clinical trials, considering real-world scenarios and
further understanding
patient outcomes. of therapeutic
Moreover, efficacy. Additionally,
refining experimental this study
protocols to encompass was conducted
a broader range un-
der specific experimental
of parameters and incorporatingconditions, andassays
functional variations in factors
could provide suchcomprehensive
a more as pH, temperature,
and ionic strength could influence results. Moving
understanding of the therapeutic potential of these mAbs. forward, it is beneficial to explore these
mAbs’ efficacy in preclinical models or clinical trials, considering real-world scenarios and
4. Materials
patient and Methods
outcomes. Moreover, refining experimental protocols to encompass a broader
® 450 mg and trastuzumab
range of parameters antibodies
The monoclonal (mAbs) pertuzumab
and incorporating functionalPerjeta
assays could provide a more compre-
Herceptin ® 150 mg (Roche Pharma AG, Basel, Switzerland),
hensive understanding of the therapeutic potential of thesetrastuzumab
mAbs. biosimilars

4. Materials and Methods

The monoclonal antibodies (mAbs) pertuzumab Perjeta® 450 mg and trastuzumab
Herceptin® 150 mg (Roche Pharma AG, Basel, Swi erland), trastuzumab biosimilars On-
Int. J. Mol. Sci. 2024, 25, 3940 10 of 14

Ontruzant® 150 mg (SAMSUNG BIOEPIS, Incheon, South Korea), and Herzuma® 150 mg
(Celltrion Healthcare, Jersey City, NJ, USA) were all obtained from University Hospital Ramón
y Cajal (Madrid, Spain) through one of the authors (J.C). The human HER2 (10004-HCCH)
extracellular domain was purchased from Sino Biological, Inc. Buffer exchange was performed
using centrifugal filter units (Amicon® Ultra-0.5 mL (Merck Millipore, Jersey City, NJ, USA).
All samples were prepared in buffer 20 mM Tris-Base ULTROL® , 150 mM NaCl, pH 7.5. The
initial concentrations of HER2 and mAbs were 0.5 and 1.5 mg·mL−1 , respectively.
Dynamic light scattering (DLS) electric field correlations were obtained for the g-
eHER2 and mAbs solutions prepared as indicates above, using the Zetasizer Nano ZS
(Malvern Instruments, Malvern, UK) at T = 309 K, equipped with disposable cuvettes
(Malvern Instruments ZEN0040). By conducting our experiments at this temperature, we
aimed to ensure that the experimental conditions closely resemble the in vivo environment
where these interactions occur. The electrophoretic mobility (EM) was measured in the
Zetasizer Nano ZS apparatus, which uses phase analysis light scattering (PALS). In this
application of the technique, a voltage is applied across a pair of electrodes placed at both
ends of a disposable capillary cell containing the particle dispersion. Disposable poly-
carbonate folded capillary cells with gold-plated beryllium–copper electrodes (Malvern
Instruments DTS1060) were used to perform the measurements. Charged particles are at-
tracted to the oppositely charged electrode, and their velocity was measured and expressed
per unit field strength as the EM, µe . Measurements were carried out in aliquots of the
mAbs stock solutions. The measurements were also performed at T = 309 K (36 ◦ C), in
samples of c = 5 mg·mL−1 . Finally, we conducted experiments utilizing DLS to investigate
the temperature resistance displayed by the mAbs. By subjecting the samples to varying
temperatures, between 293 K (20 ◦ C) to 353 K (80 ◦ C), and monitoring changes in their
hydrodynamic size using DLS, we aimed to evaluate how variations in temperature may
impact the structural integrity and stability of the mAbs studied. This temperature range
encompasses conditions relevant to both storage and potential physiological fluctuations,
thereby providing valuable insights into the thermal stability profile of the tested formu-
lations. Readers are referred to Supplementary Material for more details about the basic
characterization of the mAbs.
The analysis of the binding between mAbs and HER2 receptor and the study of
the formed complexes was conducted using SEC-TD. The SEC system used was a GPC-
TDAmax (Malvern Instruments) equipped with a Superose™ 6 increase 10/300 GL column
(Cytiva, Marlborough, MA, USA). The column was equilibrated at T = 309 K with a buffer
consisting of 20 mM Tris Base Ultrol® , 150 mM NaCl, pH 7.5. Samples of 100 µL were
injected into the SEC column and eluted with the buffer at a flow rate of 0.5 mL·min−1 . The
elution profiles were followed by UV–photodiode array (UV-PDA), differential refractome-
ter (RI), 7◦ low-angle light scattering detector (LALS), and 90◦ right-angle light scattering
detector (RALS). The data were acquired and analyzed using OmniSEC4.6 software, which
determined the absolute molecular weight, Mw ; intrinsic viscosity, [η]; specific absorption
coefficient, dA/dc; and concentration, c, of each sample. The extinction coefficient of each
species was obtained by matching the concentration measured by means of the RI detector
area to that obtained from the UV detector at 280 nm. The analysis was performed main-
taining the stoichiometry relation of the reaction at 3:1 in all cases. Three experiments were
conducted as follows: (1) HER2 was added over trastuzumab. After 30 min, pertuzumab
was added in the same proportion, and the sample was injected after an additional 30 min.
(2) HER2 was added over pertuzumab, and after 30 min, trastuzumab was added to the last
solution. Conditions and ratio of amounts added were the same as in the first experiment.
(3) A solution of trastuzumab and pertuzumab 1:1 was prepared with a concentration of
1 mg·mL−1 . Over this mixture, HER2 was added. Bovine serum albumin (Sigma Aldrich,
St. Louis, MO, USA) was used as a standard reference protein of known molecular weight,
concentration, and refractive index increment (dn/dc = 0.185 mL·g−1 ). Before each deter-
mination, a BSA solution at a concentration of 2 mg·mL−1 was used as a standard, allowing
Int. J. Mol. Sci. 2024, 25, 3940 11 of 14

for the determination of molecular weight, concentrations, intrinsic viscosity, and extinction
coefficients of the mAbs, HER2 protein, and the complexes.

5. Conclusions
In conclusion, the comprehensive characterization and comparative analysis presented
in this study shed light on the remarkable similarity between Herceptin and its biosimilars,
Ontruzant and Herzuma, across various physicochemical parameters. The findings under-
score the interchangeability of these therapeutic agents, offering promising implications for
clinical practice. Moreover, our investigation into the interactions with HER2 highlights
the equivalence of biosimilars in binding affinity, further supporting their effectiveness
in targeted therapy. The observed capacity of biosimilars to form high-order complexes,
particularly in combination with pertuzumab, underscores their potential in enhancing
therapeutic efficacy. Additionally, our study underscores the potential of coformulated
mixtures of therapeutic monoclonal antibodies to increase efficacy, offering a strategy akin
to the natural humoral immune response. These insights contribute to the growing body of
knowledge surrounding monoclonal antibody therapeutics and pave the way for future
developments in the field.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/ijms25073940/s1, refs. [45–55] cited in Supplementary Materials.
Author Contributions: Conceptualization, J.F.V., J.R. and J.C.; supervision, J.F.V. and J.C.; validation,
J.F.V., J.R. and J.C.; formal analysis, J.F.V., J.R. and V.L.C.; writing—original draft, J.F.V. and V.S.-E.;
writing—review and editing, J.F.V., J.R., V.L.C. and J.C.; project administration, J.F.V.; funding
acquisition, J.F.V., J.R. and J.C.; resources, J.C., L.G.-E., J.P.-G., M.G:, L.G., P.C., C.S., P.G. and C.O.;
investigation and methodology, E.F.-M., V.S.-E., L.G.-E., J.P.-G., M.G., L.G., P.C., C.S., P.G. and C.O.;
data curation, E.F.-M. and V.S.-E. All authors have read and agreed to the published version of
the manuscript.
Funding: This research work was funded by the Fundación FERO and Fundación “Contigo contra el
cancer de la mujer” (With you against women’s cancer). J.R. and J.F.V acknowledge support by CSIC
under the Grants PIE202050E017 and PIE202350E113.
Data Availability Statement: The data presented in this study are available upon request from the
corresponding author.
Conflicts of Interest: The authors declare that they have no known competing financial interests or
personal relationships that could have appeared to influence the work reported in this paper.

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