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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

martin.scholz@leukocare.com

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

REFERENCES

[1] D. S. Tomar, S. Kumar, S. K. Singh, S. Goswami, MAbs 2016, 8, 216.

[2] S. J. Shire, J. Liu, W. Friess, S. Jörg, H.-C. Mahler, in Formulation and Process

Development Strategies for Manufacturing Biopharmaceuticals (Eds: Jameel and

Hershenson), John Wiley & Sons, 2010, 349.

[3] S. J. Shire, Monoclonal antibodies, Meeting the Challenges in Manufacturing,

Formulation, Delivery and Stability of Final Drug Product (Ed: Shire), Woodhead Publishing

Series in Biomedicine: Number 77, Elsevier UK 2015.

[4] S. J. Shire, Curr. Opin. Biotechnol. 2009, 20, 708.

[5] J. Jezek, M. Rides, B. Derham, J. Moore, E. Cerasoli, R. Simler, B. Rerez-Jamirez, Adv.

Drug Deliv. Rev. 2011, 63, 1107.

[6] J. S. Bee, T. W. Randolph, J. F. Carpenter, S. M. Bishop, M. N. J. Dimitrova, Pharm.

Sci. 2011, 100, 4158.

[7] A. Allmendinger, S. Fischer, J. Huwyler, H. C. Mahler, E. Schwarb, I. E. Zarraga, R.

Mueller, Eur. J. Pharm. Biopharm. 2014, 87, 318.

[8] N. J. Armstrong, M. N. Bown, Y.-F. Maa, High-concentrated monoclonal antibody

formulations. Patent WO 2013/173687 A1, 2012.

[9] S. Wang, N. Zhang, T. Hu, W. Dai, X. Feng, X. Zhang, F. Qian, Mol. Pharm. 2015, 12,

4478.

[10] N. Inoue, E. Takai, T. Arakawa, K. Shiraki, Mol. Pharm., 2014, 11, 1889.

 

27

[11] R. Scherließ, A. Ajmera, M. Dennis, M. W. Carrol, J. Altrichter, N. J. Silman, M. Scholz,

K. Kemter, A. C. Marriott, Vaccine. 2014, 32, 2231.

[12] R. Tscheliessnig, M. Zörnig, E. Herzig, K. Lückerath, J. Altrichter, K. Kemter, A. Paunel-

Görgülü, T. Lögters, J. Windolf, S. Pabisch, J. Cinatl, H. Rabenau, A. Jungbauer, P. Müller-

Buschbaum, M. Scholz J. Koch, Materials today 2012, 15, 394.

[13] M. Scholz, A. Lüking, Biotechnology J. 2012, 7, 1002.

[14] S. Shimuzu, D. J. Smith, J. Chem. Phys. 2004, 121, 1148.

[15] I. P. Buzatto, A. Ribeiro-Silva, J. M. Andrade, H. H. Carrara, W. A. Silveira, D. G. Tiezzi,

Braz J. Med. Bio.l Res. 2017, 26, 50.

[16] N. Dashnor, C. R. Noe, E. Urban, B. Lachmann, Int. J.Mol. Sci. 2014, 15, 6399.

[17] W. Wang, Int. J. Pharm. 1999, 185, 129.

[18] T. Arakawa, K. Tsumoto, Y. Kita, B. Chang, D. Ejima, Amino acids, 2007, 33, 587.

[19] L. Jorgensen, S. Hostrup, E. Horn Moeller, H. Grohganz, Expert Opin. Drug Deliv. 2009,

6, 1219.

[20] J. Kang, X. Lin, J. Penerea, Bio-Process International, 2016, 14, 40.

[21] N. W. Warne, Europ. J. Pharm. and Biopharm. 2011, 78, 208.

[22] K. Kemter, K. in Biofunctional Surface Engineering (Ed: M. Scholz) Pan Stanford

Publishing Group, Singapore 2014, 11.

[23] R. M. Ionescu, J. Vlasak, C. Price, M. Kirchmeier, J. Pharm. Sci., 2008, 97, 1414.

[24] A. A. Wakankar, M. B. Feeney, J. Rivera, Y. Chen, M. Kim, V. K. Sharma, Y. J. Wang,

Bioconjugate Chem. 2010, 21, 1588.

 

28

[25] K. Diepold, K. Bomans, M. Wiedemann, B. Zimmermann, A. Petzold, T. Schlothauer, R.

Mueller, B. Moritz, J. O. Stracke, M. Mølhøj, D. Reusch, P. Bulau, PloS ONE, 2012, 7,

e30295.

[26] H. Liu, Dissertation, PhD, University of Rhode Island, April 2013.

[27] L. Khawli, S. Goswami, R. Hutchinson, Z. W. Kwong, J. Yang, X. Wang, Z. Yao, A.

Sreedhara, T. Cano, D. Tesar, I. Nijem, D. E. Allison, P. Y. Wong, Y. H. Kao, C. Quan, A.

Joshi, R. J. Harris, P. Motchnik, MAbs, 2010, 2, 613.

[28] Y. Du, A. Walsh, R. Ehrick, W. Xu, K. May, H. Liu, MAbs, 2012, 4, 578.

[29] B. K. Chavez, C. D. Agarabi, E. K. Read, M. T. Boyne, M. A. Khan, K. A. Brorson,

Biomed. Res. Int. 2016, doi: 10.1155/2016/2074149.

[30] V. Kayser, N. Chennamsetty, V. Voynov, K. Forrer, B. Helk, B. L. Trout, Biotechnology

J. 2011, 6, 38.

 

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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

 

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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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