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Research Article
Amino acid-based advanced liquid formulation development for highly concentrated
therapeutic antibodies balances physical and chemical stability and low viscosity†
Kristina Kemter
Jens Altrichter
Roland Derwand
Thomas Kriehuber
Eva Reinauer
Martin Scholz
Correspondence: Professor Dr. Martin Scholz, PhD
LEUKOCARE AG, Am Klopferspitz 19, 82152 Martinsried/Munich, Germany
Phone: +49 89 7801665-0
Fax: +49 89 7801665-11
Keywords: Medical biotechnology, Antibodies, Biotherapeutics, Chromatography, Protein
aggregation
†This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: [10.1002/biot.201700523]. This article is protected by copyright. All rights reserved Received: August 10, 2017 / Revised: March 16, 2018 / Accepted: April 3, 2018
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SUMMARY
To develop highly concentrated therapeutic antibodies enabling convenient subcutaneous
application, well stabilizing pharmaceutical formulations with low viscosities are considered to
be key. The purpose of this study was to select specific amino acid combinations that reduce
and balance aggregation, fragmentation and chemical degradation and also lower viscosity of
highly concentrated liquid antibodies. As a model, the therapeutically well-established antibody
trastuzumab (25 - >200 mg/mL) in liquid formulation was used. Pre-testing of formulations
based on a stabilizing and protecting solutions (SPS®) platform was conducted in a thermal
unfolding model using Differential Scanning Fluorimetry (DSF) and accelerated aging at 37 °C
and 45 °C. Pre-selected amino acid combinations were further iteratively adjusted to obtain
stable highly concentrated antibody formulations with low viscosity. Size Exclusion
Chromatography (SE-HPLC) revealed significantly lower aggregation and fragmentation at
specific amino acid:sugar and protein:excipient ratios. Dynamic viscosities <20 mPa*s of highly
concentrated trastuzumab (≥200 mg/mL) were measured by falling ball viscosimetry.
Moreover, less chemical degradation was found by Cationic Exchange Chromatography (CEX
-HPLC) even after six months liquid storage at 25 °C. In conclusion, specifically tailored and
advanced amino acid-based liquid formulations avoid aggregation and enable the development
of stable and low viscous highly concentrated biopharmaceuticals.
Abbreviations: CEX-HPLC, cationic exchange chromatography; SE-HPLC, size exclusion
chromatography; SPS®, stabilizing and protecting solutions
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1 INTRODUCTION
Today, there is an emerging need for highly concentrated stable therapeutic antibodies (> 100
mg/mL) in liquid formulation with low viscosity resulting in better syringeability and injectability
for convenient subcutaneous (s.c.) administration of low volumes (1-1.5 mL) [1]. The
subcutaneous route of administration is highly preferred for therapeutic indications where
home (self-) medication is desirable, for example, for chronic diseases. Subcutaneous
administration improves ease of use and avoids hospitalization for administration resulting in
increased patient compliance as well as significant reduction in workload for clinical personnel.
Thus, it contributes to reduced treatment costs for the health care system. The crucial
conflicting and challenging aspect in generating highly concentrated therapeutic antibody
formulations is to find the balance between the highest physical and chemical stability of the
protein in accordance with maximally reduced viscosity levels of the formulation [1, 2].
Highly concentrated therapeutic antibody formulations and the resulting high formulation
viscosities are associated with a particular high propensity for aggregation. Moreover, highly
concentrated therapeutic antibody formulations are further challenged by aging processes
depending on the respective storage conditions [4-6]. Along with the need for a stable highly
concentrated therapeutic antibody formulation, the final liquid formulation ought to be
characterized by low viscosity in order to enable safe, easy and painless administration [1]. In
this regard, the formulation has to be adapted to the intended route of administration.
Especially, syringeability and injectability [1, 7] are important quality features of the drug
product for subcutaneous administration [1].
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To date, different formulation strategies have been tried to reduce the viscosity of highly
concentrated monoclonal antibody formulations suitable for subcutaneous administration. As
for highly concentrated liquid formulations, the addition of special viscosity reducing excipients,
e.g. salts, amino acids or sugars to balance repulsion and attractive forces through
intermediated ionic strength or the adjustment of pH are well known strategies for viscosity
reduction during manufacturing as well as in the drug product of highly concentrated antibody
formulations [8-10]. Moreover, the desired pH for reducing viscosity or the particular desired
viscosity reducing excipient can have detrimental effects on the stability of the therapeutic
antibody [8]. Furthermore, it is well known that particularly amino acid-based formulations
containing single amino acids at high concentrations are beneficial for the stabilization of
biopharmaceuticals and the lowering of viscosities on antibody formulations up to and including
200 mg/mL [9, 10]. However, the systematic combination of amino acids has not been studied
extensively especially at concentrations >200 mg/mL. Formulations that have beneficial
stabilizing effects, e.g. related to the limitation of aggregation, may fail to avoid chemical
degradation and/or to lower viscosity. Considering these aspects, an advanced finally selected
formulation of highly concentrated therapeutic antibody formulations ought to be well-balanced
to address not only aggregation but also chemical degradation and viscosity.
Altogether, an ideal pharmaceutical formulation should be able to protect the target molecule
at different stress conditions during manufacturing, storage, and until administration of the drug
product. Recent in vitro and preclinical in vivo research revealed the efficacy of distinct amino
acid combinations in stabilizing a broad range of biomolecules even during extreme stress
conditions [11-13]. Based on the principle of preferential exclusion and preferential binding
[14], specifically tailored amino acid combinations for individual target molecules were shown
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to be highly efficient in preventing loss of molecular integrity and function in dry formulations.
For example, amino acid-based formulations enabled the stabilization of an anti-influenza A
vaccine based on Pandemrix® [11], optimum refolding during reconstitution of dried and
irradiated complex IgM antibodies [12], and retained binding specificity of anti-TNF-alpha
antibody infliximab during storage [13]. We here studied whether iterative optimization of
specific amino acid-based liquid formulations enables highly concentrated formulations of a
therapeutic antibody with sufficiently balanced stability and low viscosity.
The therapeutic antibody trastuzumab (Herceptin®) [15] was used as a model substance for
studying the molecular integrity during the application of different kinds of thermal stresses,
e.g. during thermal unfolding and during liquid storage at elevated temperatures.
Trastuzumab is a recombinant, humanized monoclonal antibody glycoprotein that selectively
targets the extracellular domain of human epidermal growth factor receptor 2 protein (Her2)
and is approved for the treatment of Her2 overexpressing breast cancer, metastatic gastric or
gastro esophageal junction adenocarcinoma [http://www.ema.europa.eu/ema/;
http://www.fda.gov/; 16]. The antibody is an IgG1 Kappa () type antibody produced in
recombinant Chinese hamster ovary (CHO) cells and contains human framework regions with
complementary-determining regions of a murine antibody that binds to Her2 [16].
Because therapeutic antibodies are particularly susceptible to the formation of aggregates,
fragmentation, and to chemical degradation [3], especially during liquid storage, the molecular
integrity was monitored by SE-HPLC and CEX-HPLC after different forced degradation
conditions and storage for up to six months at 25 °C.
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2 MATERIALS AND METHODS
2.1 Trastuzumab sample preparation
Freeze-dried (150 mg) or highly concentrated liquid (120 mg/mL; 600 mg in 5 mL) trastuzumab
(Herceptin® i.v. and Herceptin® s.c., Roche, Basel, Switzerland) were used in all experiments.
Reconstitution of freeze-dried trastuzumab in 7.2 mL water resulted in 21 mg/mL original
formulation containing 20 g/L trehalose, 0.815 g/L histidine buffer, 0.09 g/L polysorbate 20, pH
6. Highly concentrated trastuzumab contains 79.45 g/L trehalose, 3.13 g/L histidine buffer,
1.49 g/L methionine, 0.4 g/L polysorbate 20, and 0.024 g/L recombinant human hyaluronidase
(rHuPH20), pH 5.5. Reconstitution of freeze-dried trastuzumab in appropriate amounts of water
resulted in 20 mg/mL, 25 mg/mL or 50 mg/mL IgG. Liquid trastuzumab was used as original
liquid material or as concentrated liquid original material. In all other cases trastuzumab was
re-buffered using dialysis in different amino acid-based formulations (Table S1, Supporting
Information; for clarity reasons, not all used formulations shown in Table S1 are represented in
the results section) at pH 6 or pH 5.5 overnight and as controls in the original freeze-dried or
liquid formulation. Slide-A-Lyzer® dialysis cassettes (cut-off 3.5 kDa; volume 3-12 mL) were
purchased from Thermo Scientific (Darmstadt, Germany). In experiments with highly
concentrated trastuzumab formulations, an additional concentration step was done. Unless
otherwise stated, iterative modifications of formulations resulted in unchanged total amounts
(mg/mL) of excipients for control reasons.
2.2 Database driven pre-selection of pharmaceutical excipients
For excipient pre-selection, a stabilizing excipient data base (LEUKOCARE AG, Munich,
Germany) was used. The LEUKOCARE database comprises a data library with detailed
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information from literature and from past projects concerning the molecular structure of target
molecules, main degradation pathways observed in defined stress models, hot spots of
chemical and physical molecular degradation and the efficacy of individual excipients to protect
against defined stress conditions and corresponding degradation pathways. The database
structure allows for rapid retrieval of successful and regulatory compliant excipients and
formulations, specifically selected for various types of biomolecules under defined conditions
and types of preparation. For this study, the main first level research parameter „antibody“,
„IgG1“ and “trastuzumab” resulted in the identification of formulation F1 which was used for
prospective adaptation to obtain stable highly concentrated therapeutic antibody formulations.
The next level research parameters “therapeutic antibody”, “IgG1”, “thermal unfolding” and
“liquid storage” revealed pre-selected excipient combinations based on F1 as the starting point
for further adjustments within a significantly reduced design space.
2.3 Differential Scanning Fluorimetry (DSF)
DSF was performed in a real time PCR cycler (BioRad). Increasing fluorescence of the
hydrophobic dye Sypro® Orange with increasing temperature in the presence of trastuzumab
was measured. Trastuzumab stock solutions (21 mg/mL) and SyproOrange 5000 x stock
solution in DMSO were diluted in histidine buffer (pH 6.5) to 1 mg/mL and 0.75 mg/mL and 50
x Sypro® Orange. After dilution in test formulations (histidine as a control) in PCR plates (0.1
and 0.075 mg/mL trastuzumab; 5 x SyproOrange), plates were centrifuged for 1 min at 500
rpm at 4 °C. The PCR cycler was heated from 25 to 95 °C in 0.3 °C steps per minute.
Fluorescence was quantified at 490 nm excitation and 575 nm emission wavelength. Midpoints
of thermal unfolding of trastuzumab (Tm1 and Tm2) were determined after splitting of the
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measured unfolding curves into two separate sigmoidal normalized unfolding curves by fitting
of the data to the Boltzmann equation using GraphPad Prism 6.
2.4 Size exclusion chromatography (SE-HPLC)
SE-HPLC (UV-280 nm detector; UHPLC system UltiMate3000 Thermo Scientific, Germany)
and a size exclusion column TSK-gel® G3000SWXL 7.8 x 300 mm column (5 µm; Tosoh
Bioscience, Tokyo, Japan) were used at 30 °C with a flow rate of 0.5 mL/min (injection volume
25 µl). Prior to SE-HPLC, samples were diluted to 2.5 mg/mL in mobile phase (Dulbecco’s
PBS pH 7.1; PAA Laboratories, Pasching, Austria). Relative areas under the curves (% AUC)
were determined with the Chromeleon 7 Chromatography Data Software (Thermo Scientific).
2.5 Cationic exchange chromatography (CEX-HPLC)
CEX-HPLC (UV-280 nm detector; UHPLC UltiMate3000 Thermo Scientific, Germany) and a
cation exchange column TSK-gel® CM-STAT 4.5 x 100 nm (7 µm; Tosoh Bioscience, Tokyo,
Japan) was used at 45 °C and with a flow rate of 0.8 mL/min (injection volume 25 µl). Prior to
the CEX-HPLC analysis, samples were diluted to 2.5 mg/mL IgG in mobile phase A (10 mM
sodium phosphate buffer pH 7.5). The bound charge variants of trastuzumab were eluted in a
sodium chloride gradient using 0 % to 30 % buffer B (10 mM sodium phosphate buffer pH 7.5;
100 mM sodium chloride). Relative areas under the curves (% AUC) were determined with the
Chromeleon 7 Chromatography Data Software (Thermo Scientific).
2.6 Viscosimetry
Dynamic viscosity was measured as mPas*s at 20 °C by falling ball viscosimeter model AMVn
(Anton Paar, Germany). After determination of the density of a highly concentrated protein
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sample (120 mg/mL; 220 mg/mL) and calibration of the capillary with water at 20 °C using the
falling angle of 70°, the ball was introduced into the capillary and approximately 500 µl of
trastuzumab (≥ 200 mg/mL) formulations were carefully filled into the capillary which was
inserted into the capillary block of the instrument. Mean ± SD values were determined from ten
successive measurements in one filled capillary.
2.7 Statistics
All experiments were done at least in triplicates and data are depicted as Mean ± SD, except
when indicated otherwise. Differences were considered significant at p<0.05 (*), p<0.01 (**),
p<0.001 (***), p<0.0001 (****) respectively. Significances between the control buffer or original
formulation (F0), respectively and the stabilizing solutions were calculated by one-way
ANOVA. The comparison at specific time points was performed by grouped analysis by two-
way ANOVA using GraphPad Prism 6.0.
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3 RESULTS
3.1 Pre-selection of amino acid-based formulations in a thermal unfolding model using
DSF
Thermal unfolding experiments were conducted (Figure 1, Table 1) with trastuzumab (0.075-
0.1 mg/mL) using DSF analysis to characterize the stabilizing efficacy of selectively modified
amino acid-based formulations. The thermal unfolding profile of the therapeutic antibody
trastuzumab in histidine buffer at pH 7 is characterized by two midpoints of thermal unfolding
Tm1 70.3 ± 0.03 °C and Tm2 81.3 ± 0.02 °C. The initial amino acid formulation F1, comprising
seven (base) amino acids at pH 7 (Table S1, Supporting Information) was selected from our
database but exhibited only marginal effects on the midpoints of thermal unfolding (Tm1 69.1 ±
0.05 °C; Tm2 82.6 ± 0.01 °C) of trastuzumab compared to histidine buffer at pH 7 (Figure 1A;
Table 1). Specific elimination of the single amino acids arginine, histidine and lysine
responsible for the non-stabilizing effect of F1 in this thermal unfolding model and subsequent
addition of special osmolytic stabilizing compounds, e.g. trehalose significantly (p<0.001)
increased the stabilizing efficacy of the analyzed formulations F1-A to F1-C at pH 6 as shown
by the shifts of the corresponding thermal unfolding curves to higher temperatures (Figure 1B-
E; Table 1). Thermal unfolding of trastuzumab in these formulations resulted in maximum ΔTm
values of both midpoints of thermal unfolding to about 4.5-6.5 °C (formulations F1-B and F1-C
in Table 1; Figure 1D-E) compared to the thermal unfolding of the antibody in histidine buffer at
pH 6. Interestingly, when single amino acids (e.g. arginine, methionine), commonly used for
liquid storage, were used as single excipients in this model, rather destabilizing effects were
observed (not shown). However, when these amino acids were added to F1-B and F1-C,
resulting in F1-D – F1-G, stabilizing efficacy was less than in F1-B but was partially retained
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with ΔTm values about 1.3-2.7 (Table 1; Figure 1, F-I). The addition of trehalose to F1-D and
F1-F resulted in F1-E and F1-G which led to improved antibody stability with ΔTm values of
about 3.7-5.4 (Table 1; Figure 1).
3.2 Testing of pre-selected amino acid-based formulations during short-term liquid
storage at low trastuzumab concentrations
Based on the findings in preliminary liquid storage experiments that the amino acid based composition
F1 containing the seven (base) amino acids without additives resulted in remarkable chemical
changes analyzed by CEX-HPLC and to a high propensity for fragmentation with reduced aggregation
monitored by SE-HPLC iterative and specific modifications of amino acid compositions were applied
which significantly limited aggregation, fragmentation and also partly chemical degradation. In
order to determine the role of the total amount of amino acids, the different antibody:excipient
ratios, and the amino acid:sugar ratios formulations F1-1 to F1-10 were tested. Different
additives (sugars, osmolytic amino acid derivatives, antioxidants, free radical scavengers) and
combinations thereof were selected from our stabilizing excipient database and added to the
seven amino acids in F1 resulting in formulations F1-1 to F1-5 or to the four amino acids
resulting in formulations F1-6 to F1-10. Balanced concentrations and ratios between the amino
acids (7 or 4) as well as the different additives resulted in a total excipient concentration of 40
g/L and an antibody:excipient ratio of 1:1.6 (formulation F1-1 to F1-10; Table S1, Supporting
Information).
Low concentrated trastuzumab (25 mg/mL) was stored at 37 °C and 45 °C for up to 28 days in
different amino acid formulations in comparison to the original formulation. SE-HPLC analysis
at indicated time points upon the course of storage (Figure 2), revealed after 28 days of liquid
storage at 45 °C a significant increase in aggregation up to 2.5 % and fragmentation up to 2.8
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% of the antibody in the original formulation (Figure 2A). In contrast, F1-2 (seven amino acids
in combination with an antioxidant, e.g. methionine) reduced both aggregation (0.5 %) and
fragmentation (1.5 %) after 28 days at 45 °C (Figure 2B) whereas F1-6 (four base amino acids
in combination with an osmolytic amino acid derivative and trehalose) resulted in increased
aggregation (1.1 %) and fragmentation (1.6 %; Figure 2C). Particularly aggregation was
avoided by formulations F1-9 (0.4 %) and F1-10 (0.6 %; four base amino acids in combination
with an antioxidant and a free radical scavenger, respectively and trehalose; Figure 2D and E).
Because of the prominent stabilizing effects of F1-10, as observed in SE-HPLC and also CEX-
HPLC (see below), we tested different F1-10 concentrations (25, 50, and 75 mg/mL) with 25
mg/mL (not shown) and 50 mg/mL trastuzumab in order to analyze the dependence of
aggregation/fragmentation on the antibody to excipient ratio (Table S2, Supporting
Information). During liquid storage for 42 days at 37 °C, aggregation was only marginally
reduced with increasing concentration of F1-10 (0.49 %, 0.45 %, and 0.37 %, respectively),
however along with increased fragmentation (0.53 %, 0.73 %, and 0.82 %, respectively).
3.3 Further iterative optimization of amino acid-based formulations for highly
concentrated liquid trastuzumab
Trastuzumab (120, 150, and 200 mg/mL) was formulated in the iteratively adjusted formulation
F1-9 resulting initially in formulations F2-1 and F2-2 in combination with 120 mg/mL
trastuzumab (Table S1, Supporting Information) in which the amino acids:sugar (e.g.
trehalose) ratio, the amino acids:methionine ratio, and the excipients:antibody ratio were
modified and a metal chelating agent and an additional antioxidant were added. In F2-1 and
F2-2 the four base amino acids (F1-9) in a total concentration of 50 g/L were combined in F2-1
with 80 g/L trehalose, methionine and polysorbate 20 and in F2-2 with 32.2 g/L trehalose,
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methionine, polysorbate 20, a metal chelating agent and an additional antioxidant (Table S1,
Supporting Information).
Accelerated aging (Figure 3) for three months at 30 °C of the control antibody in the original
untreated liquid formulation (120 mg/mL) demonstrated a significantly (p<0.01) higher degree
of aggregation (> 0.35 %) compared to F2-1 (0 .25 %) and F2-2 (0.2 %; Figure 3A, top), in line
with formulation viscosities (Figure 4). In contrast, fragment formation during storage was
similar between the original formulation (0.4 %) and F2-1 (0.46 %), but remarkably reduced
(p<0.05) in F2-2 (0.33 %; Figure 3A, bottom). The associated decrease of the monomer peak
from the initial value of 99.8 % was limited in F2-1 to 99.3 % (p<0.01) and was even less
(p<0.01) in F2-2 (99.5 %; Figure 3A, middle) compared to the original formulation (99.24 %).
F2-2 was further modified (Table S1, Supporting Information) resulting in F2-3. Additional
alanine, a sugar mixture (trehalose/sucrose) ratio of 3:1, a moderate increase in methionine
concentration, addition of an additional antioxidant and moderate increasing concentration of
the metal chelating agent, resulted in F2-4. These formulations at increasing trastuzumab
concentrations (150 mg/mL; increased antibody:excipient ratio in the case of F2-3) further
reduced aggregation (approx. 0.5 %) and fragmentation (0.6 %; p<0.01) of the antibody during
liquid storage for 6 months at 25 °C compared to the original formulation (1 % aggregates; 1 %
fragments). The structural integrity of the antibody with an initial monomer content of approx.
99.7 % was partially retained during liquid storage for 6 months at 25 °C in F2-3 and F2-4 to
about 98.9 % compared to the highly concentrated antibody in the original formulation (98.0 %
monomers; Figure 3B).
Further modifications of F2-4 resulted in formulations tailored for 200 mg/mL trastuzumab
(Table S1, Supporting Information) with low formulation viscosity. For example, in F2-5 the
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sum of excipients was reduced from 135 g/L to 90 g/L with 1:1 amino acid:sugar ratio and 2.2:1
antibody:excipient ratio to increase antibody concentrations and to achieve low viscosities (see
below). In F2-6, the total amount of amino acids was retained while sugar was reduced
(increased amino acid:sugar ratio; 3.2:1) resulting in a further reduced amount of excipients
(increased antibody:excipient ratio). In F2-7, the same excipients were used with increased
histidine:tryptophan ratio. The trehalose:saccharose ratio was reduced to 2:1, and the amino
acid:sugar ratio was reduced to 1.5:1. The antibody:excipient ratio was comparable to F2-5
(2.2:1).
Aggregate peaks corresponding to antibody dimers (elution time ≈ 14 minutes) were
significantly reduced to 0.24 % after 3 months at 25 °C in F2-7 (p<0.01; p<0.0001), and to a
minor extent in F2-5 (0.39 %) and F2-6 (0.43 %; p<0.01, p<0.001) compared to the original
formulation during liquid storage for 3 months at 25 °C (0.63 %; Figure 3C, top). In F2-6,
(highest antibody:excipient ratio of 3.33:1) slightly stronger aggregation (0.43 %) associated
with an increase in formulation viscosity compared to F2-5 and particularly to F2-7 was found
(Figure 3C, top; Figure 4). No relevant fragmentation was observed with 200 mg/mL (Figure
3C, bottom) which might be due to the increased antibody to excipient ratios as already
observed with low concentrated formulations (as mentioned above). In all amino acid-based
formulations the aggregate peaks corresponding to dimers and the monomer peaks directly
after sample preparation comprising dialysis and concentration were comparable to the original
unstressed liquid trastuzumab formulation. In contrast, after concentration of the original
formulation a shoulder between the dimer and the monomer peak was observed suggesting
the formation of slightly higher molecular weight species than the monomer and resulted in a
higher propensity for the formation of aggregate dimers in the original formulation upon the
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whole course of storage compared to the amino acid based formulations. The monomer peak
in the amino acid based formulations was almost completely retained (initial monomer content
99.8 %) during the course of liquid storage at 25 °C (99.39 % F2-5, 99.35 % F2-6, 99.47 % F2-
7) compared to the original formulation (99.12 %; Figure 3 C, middle). F2-5 and F2-6
demonstrated the lowest increase in fragmentation (0.225 %) compared to the original
formulation (0.25 %) and F2-7 (0.24 %; p>0.05; Figure 3C, bottom).
3.4 Stabilizing amino acid-based formulations of highly concentrated trastuzumab and
low viscosities
Dynamic viscosity measurements with trastuzumab (Figures 3D and E) revealed 4.8 ± 0.06
mPa*s with 120 mg/mL (Figure 3D) and 20.5 ± 0.003 mPa*s for 220 mg/mL (Figure 3E) in the
original liquid formulation (F0). In contrast, when F2-1 and F2-2 were used for 120 mg/mL
trastuzumab, significantly lower values (p<0.0001) were obtained (3.96 ± 0.003 and 3.51 ±
0.002 mPa*s, respectively) as depicted in Figure 3D. Moreover, F2-5, and F2-7 with high
stabilizing potential particularly against aggregation for ≥ 200 mg/mL concentrations resulted in
significantly reduced (p<0.0001) dynamic viscosities for 220 mg/mL trastuzumab (15.25 ±
0.005 and 17.6 ± 0.005 mPa*s) versus 20.5 ± 0.003 mPa*s as depicted in Figure 3E.
Interestingly, F2-6 (highest antibody:excipient ratio) resulted in rather increased viscosity and
propensity for aggregation.
3.5 Iterative approach to limit chemical degradation
Chemical degradation was studied by CEX-HPLC (Figure 4). A representative CEX-HPLC
chromatogram of trastuzumab after storage for 28 days at 37 °C in formulations F1-9 and F1-
10 in comparison to the original formulation is shown in Figure 4A. A continuous increase in
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formation of acidic and basic charge variants was observed over time in most of the
formulations (Table S3, Supporting Information). However, formulations comprising seven
amino acids in combination with an anti-oxidative excipient and trehalose (F1-4) and more
pronounced in combination with a free radical scavenging excipient (F1-3 and F1-5) without
and with trehalose, resulted in a reduced formation of acidic charge variants (e.g. from 33.3 %
in F1-1; 31.1 % in F1-3, 31.6 % in F1-4 to 28.6 % in F1-5; Figure 4B; Table S3, Supporting
Information) after 28 days. Only marginal influence of the formulation additives on the
formation of basic charge variants was observed (31.9 % in F1-1, 32.5 % in F1-3, 32.8 % in
F1-4, 33.7 % in F1-5; Figure 4B; Table S3, Supporting Information), resulting in the retention of
slightly more percent AUC corresponding to the main peak species of the antibody after liquid
storage for 28 days at 37 °C. The four amino acid based formulations with osmolytic amino
acid derivatives (F1-6, -7, -8) and methionine (F1-9) were associated with the formation of a
high percentage of acidic charge variants (34.6 - 36.6 %) but also with the formation of low
percentage of basic charge variants (28.8 - 29.7 %; Figure 4B; Table S3, Supporting
Information). The addition of a free radical scavenger resulting in formulation F1-10 limited the
increase in acidic charge variants while maintaining low levels of basic charge variants even
after 28 days of storage (Figure 4B; Table S3, Supporting Information). The relative amount of
main species was only slightly increased. Similar trends were observed during liquid storage
for 7 and 14 days at 45 °C experimental stress conditions (not shown).
As shown in Table S2 (Supporting Information), the F1-10 dependent reduction in acidic
fractions was concentration dependent with only a minor increase in basic fractions, and a
stable main peak with 75 mg/mL F1-10 suggesting a relevant impact of the antibody:excipient
ratio (here: 2:1; 1:1; 1:1,5 with 50 mg/mL trastuzumab).
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As with higher concentrated (120 mg/mL) trastuzumab, F2-1 based on the iteratively adjusted
F1-9 reduced basic species (p<0.05) after liquid storage for 3 months at 30 °C (36.98 %),
confirming CEX-HPLC data with low concentrated trastuzumab in F1-9 and with basic species
comparable to the original formulation (42.4 %) in F2-2 (39.71 %) as shown in Figure 4C,
bottom. In accordance with the CEX-HPLC results of formulation F1-9 with low concentrated
trastuzumab, liquid storage for three months at 30 °C of the highly concentrated trastuzumab
in formulation F2-1 and F2-2 resulted in an increased formation of acidic species (28.1 % F2-1
and 29.5 % F2-2) compared to the original formulation (22.5 %). Thus, the loss of the main
peak from the initial value of 67-68 % was comparable to the original formulation 35.11 % in
case of F2-1 (34.93 %) and slightly more pronounced in case of F2-2 (30.8 %; Figure 4C,
middle).
After six months storage of 150 mg/mL trastuzumab at 25 °C, lower amounts of basic species
were observed for F2-3 (34.8 %) and F2-4 (34.3 %) formulations (p<0.05, p<0.01) compared to
the original formulation (35.3 %; Figure 4D, bottom). Also a slightly lesser formation of acidic
species in comparison to the original formulation (31.4 %) were observed in F2-3 (31.4 %) and
in F2-4 (32.4 %; Figure 4D, top) versus F2-1 and F2-2 compared to the untreated original
formulation in the previous experiment (120 mg/mL trastuzumab; Figure 4C, top). The main
peak relative AUC was slightly more stable, particularly for F2-3 versus the original formulation,
e.g. after liquid storage for 42 days as well as 84 days at 25 °C (Figure 4C, middle).
Formulation of highly concentrated trastuzumab (200 mg/mL) with further iteratively adjusted
formulations F2-5 – F2-7 and subsequent liquid storage for 3 months at 25 °C resulted in
increased formation (not significant) of acidic charge variants (22.6 %; lowest degree in F2-7;
Figure 4E, top) but less formation of basic charge variants particularly in the case of F2-5 (32.3
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%) and F2-6 (32.2 %; p<0.05) compared to the original formulation (37.5 %; Figure 4E,
bottom). Thus, the loss of the main peak area during liquid storage for 3 months at 25 °C of the
highly concentrated antibody was partly prevented by the formulations F2-5 to F2-7 and was
comparable to the original formulation (Figure 4E, middle). In formulation F2-7 the loss of the
main peak was slightly more pronounced compared to the other formulations. The w/w ratio
between the two selected amino acids tryptophan and histidine was changed iteratively
between formulations F2-1 to F2-7 and resulted in modified formation of acidic and basic
charge variants (Figure 4E, top and bottom).
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4 DISCUSSION
In the present study we demonstrate that amino acid-based advanced formulation
development enables ideal balancing between high physical and chemical stability of
therapeutic antibodies in low viscous liquid formulations even at concentrations of ≥ 200
mg/mL. Amino acids are commonly used as excipients in protein formulations [3, 17-20], alone
or in combination with sugars or sugar alcohols. Currently, approved therapeutic antibody
liquid formulations may comprise a) histidine as buffering agent, b) arginine to avoid
aggregation, c) glycine as osmolytic stabilizer, bulking agent and/or tonicity adjusting agent,
and d) methionine as an antioxidant. In addition, the amino acid salts arginine*HCl, lysine*HCl,
histidine*HCl and sodium glutamate have recently been reported to reduce viscosity in highly
concentrated antibody solutions up to 200 mg/mL in conjunction with reduced aggregation [3,
9, 10].
However, systematically tailored adaptation of highly concentrated low viscous antibody
formulations by means of rational and stepwise combination of amino acids as main excipients
alone or in combination with other excipients to ensure maximum stability under distinct stress
conditions has not been reported yet. Moreover, the systematic balancing of amino acid
combinations, amino acid:sugar ratios and antibody:excipient ratios in this study differs from
the common, rather empiric addition of single amino acids to commercial formulations [3, 20,
21].
As a starting point, we selected a previously elaborated amino acid-based formulation which
protected dry trastuzumab under extensive thermal and irradiation stress [22] from our
database. We anticipated that the same formulation may also stabilize trastuzumab during
thermal stress and liquid storage even at higher antibody concentrations. But surprisingly, this
20
formulation (here: F1), was insufficient under these conditions. Therefore, we proposed the
general need for stress specific tailoring of amino acid formulations.
The initial iteration round with trastuzumab based on F1 was conducted in a thermal unfolding
model. The data confirmed the published characteristic thermal unfolding profile of
trastuzumab with two transition temperatures which are related to the unfolding of the antibody
CH2 region as well as the CH3 region and Fab region, respectively [23, 24].
Elimination of single amino acids (arginine, histidine and lysine) and subsequent addition of
different osmolytic, stabilizing compounds, e.g. trehalose resulted in gradually increasing shifts
of both midpoints of thermal unfolding to higher temperatures (F1-A to F1-C). The evaluated
comparable shifts of the two transition temperatures by the stabilizing effect of the selected
amino acid mixtures in combination with the added osmolytic stabilizing compounds,
suggested a particular stabilizing osmolytic effect on the whole antibody molecule (CH2
domain, CH3 domain and Fab fragment) during thermal unfolding according to the preferential
exclusion theory [14]. Interestingly, arginine and methionine (known stabilizers in liquid
therapeutic protein formulations) alone destabilized trastuzumab in our unfolding model (not
shown) which was not observed when they were added to F1-A,-B, -C formulations, suitable
for liquid storage. We therefore concluded that the formulation preselection procedure in the
thermal unfolding model is important for further adjustments to address liquid storage
requirements (e.g. chemical changes play a minor role in the unfolding model but a major role
in liquid storage).
Indeed, specific modifications of F1 (high propensity for fragmentation but low aggregation) by
varying the total amount of amino acids, antibody:excipient ratios, and amino acid:sugar ratios
limited aggregation, fragmentation, and partly avoided chemical changes. The resulting
21
antibody degradation profiles upon short-term storage of low concentrated trastuzumab in our
study matched well with published degradation profiles of trastuzumab such as asparagine
deamidation, asparagine, aspartate isomerization and methionine oxidation resulting in
aggregation and fragmentation during prolonged storage [16]. This general observation leads
to the suggestion that the accumulated chemical changes in the antibody during short-term
liquid storage may trigger aggregation during prolonged storage [16]. In accordance with the
published degradation of trastuzumab during short-term liquid storage for up to 28 days at 45
°C [16], our study revealed an increasing tendency for the formation of aggregates and
fragments over time particularly in the original formulation. Iterative adjustment of the amino
acid-based formulations especially with antioxidants and radical scavengers resulted in specific
modulations of aggregation and fragmentation. Thus, the induction of strong structural
changes in form of aggregates during prolonged liquid storage [16] can be avoided by early
adaptation of the formulation. The underlying mechanisms might be due to intermolecular
disulfide-crosslinking (oxidation) either of a small amount of unpaired cysteine residues in
recombinant antibodies or after reduction of an existing disulfide bond and rearrangement of
the two resulting unpaired cysteine residues in another disulfide bond (oxidation) between two
antibody molecules [3]. Accordingly, formulations F1-6 to F1-8 resulted in increasing aggregate
formation (antibody dimers) which was avoided in the presence of antioxidants and radical
scavengers (F1-9, F1-10).
The observed continuous increase of acidic and basic species during liquid storage at 37 °C
for up to 28 days in all analyzed formulations most likely were due to deamidation (acidic
species) and aspartate isomerization of asparagine 30 and 55, respectively and isomerization
of aspartate 102 (basic species) [25] and the oxidation of the methionine residues 255 and 431
(basic species) in the Fc region of trastuzumab [3]. Importantly, we found that the addition of
22
an antioxidant, e.g. methionine, and more pronounced of a radical scavenger (Maillard reaction
inhibitor) reduced the formation of acidic charge variants and retained the amount of main
species, however without limiting the formation of basic charge variants upon liquid storage for
up to 28 days at 37 °C. At pH 6.0 of the formulation, the hydrolysis of glycation adducts during
liquid storage might result in increasing amounts of free glucose molecules and partly to
unappreciated advanced glycation products by Maillard reaction [26]. We assume that the
addition of radical scavengers and Maillard reaction inhibitors to the mixture of 7 base amino
acids probably might have been reduced the generation of advanced glycation products,
entailing decreased amounts of acidic species in F1-3 and F1-5 [27, 28]. In addition, the basic
amino acid lysine*HCl a component of the 7 base amino acids in F1-1 to F1-5, might have
protected trastuzumab by reacting with the hydrolysis product glucose thus avoiding the
Maillard reaction between glucose and a lysine residue in the protein. This would explain the
increasing acidic charge variants in formulations F-1-6 to F1-9 after elimination of lysine*HCl
[26-28].
We could also show that the inverse concentration ratio of histidine buffer to tryptophan in
combination with an additional radical scavenger and antioxidant limits the generation of basic
fractions, probably by preventing methionine oxidation during liquid storage of the antibody [3,
27, 28]. Defined modifications of tryptophan:histidine ratios thus might generally be helpful to
specifically protect proteins from damages related to methionine oxidation.
To fulfill the stability and viscosity requirements of highly concentrated liquid trastuzumab
during long-term storage experiments, the adjustment of the amino acid:sugar, amino
acid:methionine, antibody:excipient ratios and the optional addition of metal chelators and
antioxidants resulted in significantly reduced aggregation and reduced viscosities during
23
storage for three months at 30 °C. As protein-protein interactions result in increasing
viscosities along with increasing protein concentrations, the minimized aggregation may be the
reason for the retained low viscosities. The molecular mechanisms underlying the observed
reduced aggregation and fragmentation during liquid storage for 3 months at 30 °C might be
similar to the mechanisms found with low concentrated trastuzumab (Table S1, F1-9).
However, pH adjustment with HCl instead of citric acid and the modification of the
histidine:tryptophan ratio (3 and 6 months at 25 °C, respectively) further improved stability at
low viscosity.
Interestingly, fragmentation was only a minor event during liquid storage of highly concentrated
trastuzumab formulations probably due to the increasing antibody to excipient ratio. Our
experiments revealed more aggregation and simultaneously less fragmentation with increasing
antibody to excipient ratio. Thus, a balanced antibody to excipient ratio should be considered
for stable highly concentrated antibody formulations associated with low viscosities.
Because of the theoretically rather unlimited number of possible excipient combinations as well
as tailored ratios between selected excipients, our amino-acid biopharmaceutical formulation
database was used for excipient pre-selection. In contrast to common screening methods that
mostly result in histidine or phosphate buffers for pH adjustment, together with other excipients
to address stability of therapeutic antibodies during standard storage conditions [20,21], our
pre-selection phase starts with advanced knowledge about excipient combinations that
previously were shown to limit specific degradation pathways at hot spots of chemical and
physical molecular changes. Because of our large datasets for retrospective screening of
successful and highly specific stabilizing excipients and formulations in combination with
literature-known characteristic stabilization data of the target molecule, multifold distinct
24
aspects such as aggregation, fragmentation, chemical modification, viscosity, and functionality
at defined stress conditions are easily surveyed. In combination with advanced statistical tools,
this database significantly increases the predictability of stabilizing formulations by systematic
pre-selection steps. Subsequent case-by-case adaptation steps, e.g. by DoE approaches [29]
with small design spaces, rapidly enable the ideal balancing of chemical and physical stability
of the target molecule even at high concentrations in low viscous liquid formulations. Future
studies with support of our database, for example, will elucidate the herein observed
interesting effects of amino acid excipients on antibody glycosylation [30].
In conclusion, our data are of significant importance for the manufacturing of stable highly
concentrated therapeutic antibodies because they clearly indicate that advanced amino acid
combinations protect from distinct stress-mediated molecular damages and ensure low
viscosities.
25
Acknowledgements
We thank Ivana Djordjevic and Sabine Kietz for excellent technical assistance.
Conflict-of-interest
At the time of the study all authors were employees of LEUKOCARE AG, Martinsried,
Germany.
26
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29
Table 1: Thermal midpoint values measured by DSF.
Formulation Tm1
Mean ± SD
ΔTm1 Tm2
Mean ± SD
ΔTm2
Buffer pH 7 70.3 ± 0.03 81.3 ± 0.02
F1 pH 7 69.1 ± 0.05 -1.2 82.6 ± 0.01 1.3
F1-A pH 7 72.5 ± 0.03 2.2 84.9 ± 0.05 3.6
Buffer pH 6 66.0 ± 0.06 79.5 ± 0.05
F1-A pH 6 69.7 ± 0.04 3.7 83.0 ± 0.01 3.5
F1-B pH 6 70.4 ± 0.05 4.4 83.9 ± 0.01 4.4
F1-C pH 6 72.5 ± 0.16 6.5 85.7 ± 0.02 6.2
F1-D pH 6 68.7 ± 0.07 2.7 82.0 ± 0.04 2.5
F1-E pH 6 71.4± 0.04 5.4 83.9 ± 0.03 4.4
F1-F pH 6 67.4 ± 0.02 1.4 80.8 ± 0.03 1.3
F1-G pH 6 70.2 ± 0.09 4.2 83.2 ± 0.04 3.7
Tm1 and 2: midpoint of thermal unfolding; SD: standard deviation
30
Figure legends
Figure 1. Differential Scanning Fluorimetry (DSF) with liquid trastuzumab formulations.
Thermal unfolding profiles (two thermal transition midpoints Tm1 and Tm2) of trastuzumab were
shown for amino acid formulations (blue) versus histidine buffer (red), (A) in the presence of
formulation F1; (B) at pH7 and (C) at pH6 in F1-A (without arginine, lysine, histidine, plus
osmolytic glycine derivative); (D) in F1-B (= F1-A with osmolytic alanine derivative instead of
osmolytic glycine derivative); (E) in F1-C (= F1-B plus trehalose); (F) in F1-D (= F1-A plus
methionine instead of osmolytes); (G) in F1-E (= F1-D plus trehalose); (H) in F1-F (= F1-D plus
arginine); (I) in F1-G (= F1-F plus trehalose). Statistically significant differences are indicated
by the respective P values in each graph.
Figure 2. Size exclusion chromatography (SE-HPLC) analysis of trastuzumab during
liquid storage. Chromatograms after 21 days (t =21) and 28 days (t = 28) at 45 °C of 25
mg/mL trastuzumab compared to day 0 (t = 0), formulated (A) in the original supplier
formulation of the freeze-dried product and in the amino acid-based formulations; (B) F1-2 (7
amino acids and antioxidant, e.g. methionine); (C) F1-6 (4 amino acids; trehalose and an
osmolyte); (D) F1-9 (4 amino acids; trehalose and an antioxidant, e.g. methionine) and (E) F1-
10 (4 amino acids; trehalose and a radical scavenger). The relative AUC for aggregates,
monomers and fragments are provided in the tables.
Figure 3. SE-HPLC analysis of highly concentrated trastuzumab during liquid storage
and dynamic viscosities (mPa*s). Relative AUC of aggregate peaks (top), monomer peaks
(middle) and fragment peaks (bottom) obtained by SE-HPLC are depicted. (A) Relative AUC of
SE-HPLC peaks of 120 mg/mL trastuzumab during liquid storage for 3 months at 30 °C in F2-1
and F2-2 compared to the original formulation. (B) Relative AUC of SE-HPLC peaks of 150
mg/mL trastuzumab during liquid storage for 6 months at 25 °C in F2-3 and F2-4 compared to
the original formulation. (C) Relative AUC of SE-HPLC peaks of 200 mg/mL trastuzumab
during liquid storage for 3 months at 25 °C in F2-5, F2-6 and F2-7 compared to the original
liquid formulation. Original: (120 mg/mL) F0 formulation (F0). (D) Dynamic viscosities of the
highly concentrated trastuzumab formulations (120 mg/mL) after re-buffering in formulation F2-
1 and F2-2 using dialysis compared to the untreated liquid trastuzumab product. (E) Dynamic
viscosities of the highly concentrated trastuzumab formulations (220 mg/mL). For control
31
purposes the original supplier formulation F0 was concentrated to reach 220 mg/mL.
Statistically significant differences between data groups are indicated as *p<0.05, **p<0.01,
**p<0.001, ****p<0.0001; n.s., not significant.
Figure 4. Cationic exchange chromatography (CEX-HPLC) analysis of trastuzumab
during liquid storage. (A) Representative CEX-HPLC chromatograms of 25 mg/mL
trastuzumab after liquid storage for 28 days at 37 °C in F1-9 and F1-10 compared to the
original supplier formulation of the freeze-dried product. The charge variants of the antibody
are eluting overtime in the order acidic fractions, main peak, and basic fractions. (B) The acidic
(red bars) and basic fractions (blue bars) from 25 mg/mL trastuzumab after liquid storage for
28 days at 37 °C in F1-4; F1-5 (7 amino acids); F1-9 and F1-10 (4 amino acids) revealed less
acidic fractions in the presence of a radical scavenger (F1-5 and F1-10) and in addition almost
retained limited basic fractions in the corresponding formulations containing 4 amino acids (F1-
10) in comparison to F1-5 and F6-9. (C) Relative AUC of CEX-HPLC peaks of 120 mg/mL
trastuzumab during liquid storage for 3 months at 30 °C in F2-1 and F2-2 compared to the
original formulation. (D) Relative AUC of CEX-HPLC peaks of 150 mg/mL trastuzumab during
liquid storage for 6 months at 25 °C in F2-3 and F2-4 compared to the original formulation. (E)
Relative AUC of CEX-HPLC peaks of 200 mg/mL trastuzumab during liquid storage for 3
months at 25 °C in F2-5, F2-6 and F2-7 compared to the original liquid formulation. Original:
(120 mg/mL) F0 formulation (F0). (C-E) acidic species (top), main peak species (middle) and
basic species (bottom).
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