International Journal of Pharmaceutics 436 (2012) 282–290

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International Journal of Pharmaceutics

journal homepage: www.elsevier.com/locate/ijpharm

Long-term stability of diluted solutions of the monoclonal antibody rituximab

Muriel Paul ∗ , Victoire Vieillard, Emmanuel Jaccoulet, Alain Astier
APHP, GH Henri Mondor, Département de Pharmacie, Unité Pharmaceutique de Recherche en Essais Cliniques, 51 avenue du Maréchal de Lattre de Tassigny, 94010 Créteil, France

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

Article history: As it is an important challenge for pharmacists to access the stability of monoclonal antibodies because
Received 23 February 2012 of the widespread of centralized preparation units, we conducted a study to evaluate the physicochem-
Received in revised form 29 June 2012 ical and biological stability of diluted rituximab at 1 mg/mL over six months at 4 ◦ C. We also conducted
Accepted 30 June 2012
the study at 40 ◦ C to demonstrate that all methods employed were stability indicating. Various protein
Available online 10 July 2012
characterization methods were used to determine changes in physicochemical properties of rituximab,
including size-exclusion chromatography, dynamic light scattering, turbidimetry, cation-exchange chro-
Keywords:
matography, second-derivative ultraviolet and infrared spectroscopy, and peptide mapping. Cell culture
Rituximab
Physicochemical stability
was used to assess biological stability. We demonstrated that diluted rituximab stored at 4 ◦ C in polyole-
Biological stability fine bags remained stable for at least six months. No physical or chemical instability was observed, and
Diluted solution the biological activity was fully maintained. Size exclusion chromatography did not show polymerization
Monoclonal antibody or fragmentation. No difference was noticed in the hydrodynamic diameter of RTX. No additional peak or
decrease in the areas under curve was found by cation exchange chromatography. The thermal aggrega-
tion curves and their derived thermodynamic parameters were unchanged. The primary, secondary and
tertiary structures of the protein were not modified. Finally, the direct cytotoxic effect of rituximab was
fully maintained.
© 2012 Elsevier B.V. All rights reserved.

1. Introduction Many of these stress factors are routinely encountered during

Monoclonal antibodies (mAbs) are a class of biopharmaceuti-
cal products mostly used as therapeutic agents. They represent
the fastest-growing segment of the biopharmaceutical market
(Nicolaides et al., 2006). Rituximab (RTX), the first IgG1 mAb suc-
cessfully used in onco-hematology, is a chimeric mAb indicated in
Non-Hodgkin’s Lymphoma (Cheung et al., 2007), rheumatoid pol-
yarthritis (Edwards et al., 2004) and chronic lymphoid leukemia. It
is directed against CD20, which is present in all the B-cell lines, and
induces a depletion of CD20-positive cells (Pedersen et al., 2002).
Because of its proteic nature, RTX may go through a variety of
chemical and physical degradation processes, as recently reviewed
by Manning (Manning et al., 2010). Physical instability mostly con-
cerns low energy bonds (such as hydrogen bonds), and includes
adsorption on to surfaces, unfolding and aggregation (Wang, 1999;
Chi et al., 2003; Wang et al., 2007). Chemical instability, such as
asparagine deamidation (the most common chemical degradation
process), aspartic acid isomerization, oxidation or disulfide bond
shuffling, concerns covalent bond modifications (Wakankar and
Borchardt, 2006; Chumsae et al., 2007).
∗ Corresponding author at: Département de Pharmacie, GH H. Mondor, 51 avenue
du Maréchal de Lattre de Tassigny, 94010 Créteil, France. Tel.: +33 149812760.
E-mail address: muriel.paul@hmn.aphp.fr (M. Paul).
the preparation, purification, shipping or storage of protein prod-
ucts (Jiskoot et al., 1990). Therefore, careful control of these factors
helps preserve the stability of the protein, which is a real challenge
for pharmaceutical manufacturers. Additionally, the preparation
must be stabilized against deleterious environmental factors to
preserve its pharmacological properties and to reduce potential
immunogenicity (Schellekens, 2002; Hermeling et al., 2004; Hwang
and Foote, 2005; Sharma, 2007a,b).
RTX is formulated as a 10 mg/mL solution containing sodium cit-
rate dihydrate as buffer (pH 6.5) (Wang et al., 2007) and surfactant
polysorbate 80. Their thermodynamic and physicochemical prop-
erties favor the equilibrium of attractive and repulsive strengths
and thus avoid aggregation (Chi et al., 2003). The use of citrate
buffer slows the deamidation rates by altering the conformation
of the protein (Zheng and Janis, 2006).
However, according to the manufacturer’s instructions, RTX
must be diluted into a solution of 0.9% sodium chloride and admin-
istered within 24 h. Therefore, in practice, the stability of diluted
RTX would be only 24 h. There is surprisingly little information on
mAbs’ storage stability, except for some therapeutic immunoglob-
ulins that exhibit a long shelf life (over one year) in liquid state.
Moreover, Bakri reported in 2006 that bevacizumab was stable for
a period of six months at 4 ◦ C (Bakri et al., 2006). Thus, it can be
hypothesized that this recommended short-term stability is mainly
focused on avoiding bacterial contamination, for handling in wards
(Bardin et al., 2011).

0378-5173/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ijpharm.2012.06.063
 M. Paul et al. / International Journal of Pharmaceutics 436 (2012) 282–290
It is an important challenge for pharmacists to assess the stabil-
ity of biotechnology-derived anticancer drugs such as antibodies
because of the widespread, ready-to-use, centralized preparation
of these drugs in pharmacy departments. Thus, the objective of this
study was to assess the physical, chemical and biological stability
of diluted RTX (1 mg/mL) over six months under an optimal storage
temperature (Bardin et al., 2011).
As mAbs are more susceptible to degradation at 40 ◦ C than 4 ◦ C
(Liu et al., 2006), we also conducted experiments at 40 ◦ C to deter-
mine the different instability mechanisms and to demonstrate that
the methods used were good indicators of protein stability.
Various protein characterization methods were used to deter-
mine changes in physicochemical properties of RTX, including
size-exclusion chromatography (SEC), dynamic light scatter-
ing (DLS) and turbidimetry, cation-exchange chromatography
(CEX), second-derivative ultraviolet and Fourier-transform infrared
spectroscopy (FT-IR), and peptide mapping after trypsin and
endopeptidase digestion. Cell culture was used to assess biological
stability.
2. Materials and methods
2.1. Reagents
Disodium hydrogen phosphate, sodium azide, potassium dihy-
drogen phosphate and trifluoroacetic acid (TFA) were purchased
from Merck (Darmstadt, Germany). Ammonium bicarbonate, ace-
tonitrile, dithiothreitol (DTT), guanidine hydrochloride (GnHCl),
iodoacetic acid, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-
tetrazolium-5-carboxanilide, sodium salt (XTT), and phenazine
methyl sulfate (PMS) were obtained from Sigma–Aldrich (St Louis,
MO, USA). Formic acid was purchased from Prolabo (Fontenay-
sous-Bois, France). Isotonic saline was provided by Fresenius Kabi
(Louviers, France). All reagents were of analytical grade.
2.2. Materials
RTX solutions were obtained from the commercially avail-
able product Mabthera® (Roche, Switzerland), formulated in
7.35 mg/mL sodium citrate dihydrate, 9 mg/mL sodium chloride,
sodium hydroxide and hydrochloric acid to obtain a pH of 6.5, and
polysorbate 80 (PS80) as a stabilizing agent. The formulation was
presented in glass vials as a concentrated solution of 10 mg/mL
ready to dilute in 0.9% sodium chloride solution, according to the
manufacturer’s instructions.
2.3. Methods
For this study, RTX diluted bags were prepared from Mabthera®
(Roche Laboratories) by dilution with 0.9% sodium chloride in
Freeflex® polyolefin bags (Freeflex® - Fresenius Kabi, France). The
final concentration was 1 mg/mL. Two different storage temper-
atures were tested: 4 ◦ C [2–8 ◦ C] and 40 ◦ C in an ICH climatic
incubator (60% of relative humidity Climacell® ; Bioblock Fisher Sci-
entific, Illkirch, France). For each temperature, three batches were
prepared, and all RTX diluted bags were protected against light with
opaque plastic bags.
2.4. Samples preparation
Samples were withdrawn from diluted RTX bags under sterile
conditions in a laminar flow hood to prevent potential bacterial or
particulate contaminations at days D0, D14, D30, D90 and D180.
A reference sample was obtained from the immediate dilution
of fresh RTX vials at 1 mg/mL, and was freshly prepared before
each step. The samples were centrifuged at 5000 rpm for 5 min
283
(Sigma® centrifuge; Osterode, Germany) before SEC, CEX and UV
spectrophotometric analysis. All buffers were filtered at 0.22 ␮m
and all dilution steps were conducted under laminar airflow.
2.5. Turbidity determination (Mahler et al., 2005)
The turbidimetric method is often used to estimate the amount
of visible protein aggregates by measuring the optical density of
samples based on light scattering in the near-UV or visible regions
(Thirumangalathu et al., 2006). Aggregation of RTX was monitored
by measuring absorbance at 350 nm (Seefeldt et al., 2005) using a
UV–VIS spectrophotometer (Cary 50 Probe, Varian, Inc., Palo Alto,
USA). Here, the turbidance (T ) was measured as absorbance at
350 nm, where none of the known intrinsic chromophores in the
protein formulation absorb (Wang, 2005).
The samples were diluted to 0.4 mg/mL in buffer (20 mM
KH2 PO4 , pH 6.0). The mean value of each experimental sample
absorbance was calculated and compared to the mean value of the
reference sample.
2.6. Tertiary structure analysis (Balestrieri et al., 1980)
The tertiary structure of RTX was examined by second deriva-
tive UV spectroscopy. This method allows for an evaluation of the
impact of dilution and storage on the aromatic amino acids of
the mAb. Indeed, the position of second derivative UV absorbance
peaks is sensitive to the polarity of the microenvironment of the
aromatic amino acids and therefore can be used to provide a global
picture of the tertiary structure of a protein. A Cary 50 Probe
UV–visible spectrophotometer (Varian) was used. The spectra were
collected in the near-UV region from 320 to 250 nm in a 1 cm
path length quartz cuvette. Five major peaks were studied (252-
Phe, 258-Phe, 275-Tyr, 284-Thy/Trp and 292-Trp), as previously
reported for IgG2, and also two minima at 287 and 295 nm (Servillo
et al., 1982; Kueltzo et al., 2007). The spectra of each sample (D30,
D90 and D180) were compared to the spectra of reference sample.
2.7. Determination of molecular hydrodynamic diameters
Dynamic light scattering (DLS) is a sensitive method to evaluate
the size of aggregate populations containing aggregates of 1 nm
to 6 ␮m. The analysis is based on the measurement of fluctuating
scattered light intensity due to Brownian particles motion, which
also defines diffusion.
A Malvern Zetasizer Nano ZS (Worcestershire, UK) laser light
scattering system was used to obtain DLS measurements with a
633 nm laser source. The NanoZS® software was used for data
acquisition and analysis. A total of 400 ␮l of each sample at
1 mg/mL was added in a 1 cm path length quartz microcuvette
rinsed before use with 0.22 ␮m filtered water. All the operations
were performed under laminar airflow. The size and hydrodynamic
diameter distributions of the detected aggregates were obtained by
the measurement and integration of the light scattering intensity.
2.8. Thermal denaturation curves (Wang, 1999; Harn et al., 2007)
With increases in temperature or simply with time, the sec-
ondary, tertiary and quaternary structures of a protein may change
and lead to protein unfolding and/or aggregation, which are crit-
ical factors of physical instability. These events can be monitored
because dramatic changes in protein size during denaturation can
be easily identified with DLS techniques. We selected a heating
rate of 1 ◦ C/min to draw the heating curves, allowing the system
to be a quasi-thermodynamic equilibrium showing good repeti-
tion of the results. So, the automated measurements were collected
using a 1 ◦ C incremental temperature ramp and a three-minute
284 M. Paul et al. / International Journal of Pharmaceutics 436 (2012) 282–290
equilibrium time at each temperature measurement from 25 ◦ C to
90 ◦ C. The temperature of aggregation (Tagg ), at which the aggre-
gated fraction is 0.5 during thermal transition, can be determined
using the nonlinear least squares method to fit the diameter depen-
dence on the temperature. The thermodynamics parameters were
calculated as previously by Farruggia et al. (1997).
2.9. Secondary structure analysis (Dong et al., 1997)
The secondary structure of RTX was monitored by second
derivative FT-IR spectroscopy in the conformation-sensitive amide
I band region (1600–1700 cm−1 ), which is due almost entirely to
C O stretch vibrations of peptide linkages (Kong and Yu, 2007).
This method has been extensively used in protein denatura-
tion/aggregation studies (Wang, 1999). The samples (D0, D30, D60
and D180 at 4 ◦ C) were concentrated to 16 mg/mL using 10 kDa
Centricon concentration devices (Millipore, Molsheim, France). IR
spectra were recorded with a 2000 FT-IR system (Perkin-Elmer,
les Ulis, France). For each spectrum, a 256-scan interferogram was
collected in single-beam mode, with a 4 cm−1 resolution. The sam-
ple cell consisted of CaF2 windows separated by a Teflon spacer of
15 ␮m. The spectrum of reconstituted buffer (identical composition
to the one used in the commercialized drug) was obtained under
identical conditions. For the analysis, the spectra were initially con-
verted to absorbance followed by subtraction of the corresponding
buffer spectrum. Second-derivative spectra were calculated with
the Spectrum® software (Perkin-Elmer) to calculate peak areas
and determine the percentage area under each peak. All spectra
obtained were the mean of three separate scans. The absorbance
wavelengths for the different secondary structures were those
described by different authors (Dong et al., 1997; Matheus et al.,
2006; Kong and Yu, 2007).
2.10. Chromatographic analysis (Lahlou et al., 2009)
All chromatographic analyses were realized with a Dionex
Station consisting of a Dionex Ultimate 300 chromatographic sys-
tem coupled with a Chromeleon® chromatography workstation
(Dionex Corporation, Sunnyvale, CA, USA). The system is equipped
with a gradient pump with degas option and gradient mixer, UV
detector and autosampler.
2.10.1. Cation exchange chromatography (CEX)
The different ionic species of RTX were separated on a Dionex
Propac® WCX-10 cation exchange column (4 mm × 250 mm) with a
WCX-10 guard column (4 mm × 50 mm, Dionex, Voisins le Breton-
neux, France). Buffer A was prepared with 50 mM KH2 PO4 , 0.05%
NaN3 pH 6.0, and buffer B was prepared with 50 mM KH2 PO4 , 1 M
sodium chloride and 0.05% NaN3 (w/v), both in deionized water.
The flow rate was 1 mL/min, and the injected volume was 100 ␮L.
The elution gradient started with 100% of A to achieve 50% of A and
B after 20 min, followed by an equilibrium phase of 5 min to obtain
100% of buffer A. The elution was monitored at 280 nm. The mean
value of the area under the curve (AUC) and the retention time (rt)
were compared to those of the reference sample.
2.10.2. Size-exclusion chromatography (SEC)
SEC analysis is a classical method used in protein studies to
detect potential fragmentation and aggregation (small size aggre-
gates), corresponding to physical and chemical instabilities. A
7.8 mm × 300 nm TSK-GEL G4000 SWXL column (Tosoh Biosciences
Corporation, Montgomeryville, PA) with a guard column was used,
and the elution was monitored at 280 nm. A total of 100 ␮L of each
centrifuged sample (1 mg/mL) were eluted isocratically with a pH
7.0 buffer [0.1 M sodium sulfate, 0.1 M sodium hydrogen phosphate
and 0.05% sodium azide (w/v)], at a flow rate of 0.6 mL/min. The
temperature was set at 25 ◦ C. The parameters studied were the AUC,
the retention time and the molecular weight.
The molecular weight of the peaks was calculated using a stan-
dard solution including several molecular weight markers ranging
from 1.2 to 150 kDa (hydroxocobalamin, cytochrome C, albumin
and cetuximab), and using a calibration curve fitted as a third-
degree polynomial function.
2.10.3. Peptide mapping (Dick et al., 2009)
All samples were concentrated at 16 mg/mL using Amicon®
Ultra-10 centrifugal filter units (Millipore) by centrifugation at
4000 × g for 20 min. One milliliter of the concentrated sample was
withdrawn for the next steps. These steps are identical to those
described par our team in a previous article (Lahlou et al., 2009) and
consisting in denaturation and trypsin and endopeptidase diges-
tion.
2.11. Direct cytotoxicity assay (Yang et al., 2003)
To detect potential changes in RTX biological activity after
long-term storage, RTX direct cytotoxic (intrinsic toxicity) on
CD20-expressing cells was determined through concentration-
dependent cytotoxicity curves on a human lymphoma cell line.
RAJI lymphoma cells (ATCC number: CCL-86) were cultured in
dye-free RPMI 1640 medium supplemented with 10% fetal bovine
serum and 1% glutamine at 37 ◦ C in a humidified CO2 incubator (5%
CO2 ). After dilution at 0.5 × 105 cells/mL, 100 ␮L aliquot of the cell
suspension were transferred to each well of a 96-well plate and
cultured with various concentrations of RTX (0, 4.5, 9, 22.5, 45, 90,
135, 270 and 450 ␮g/mL) for three days. Twelve wells were seeded
for each concentration tested. To assess cell viability, 50 ␮L of a
solution containing 50 ␮g of XTT and 0.38 ␮g of PMS were added.
The cells were incubated at 37 ◦ C for 4 h. The absorbance of each
well at 450 nm was measured using an automated plate reader.
The results were expressed as the percentage of inhibition in com-
parison to control (without RTX). The IC50 and IC25 were calculated
using the Michaelis–Menten model. The AUCs of the cytotoxicity
curves were also calculated, using the trapezoidal method.
2.12. Statistical analysis
The results are presented as mean ± standard deviation (SD)
obtained from three separate samples and for each storage con-
dition and concentration. All data were analyzed with the Kyplot
software (V2. Koichi Yoshioka). Analysis of variance (two-way
ANOVA) or non-parametric Mann–Whitney tests, if required, were
used to compare data. Statistical significance was defined as
p < 0.05.
3. Results
3.1. Turbidity
No variation in optical density was observed for six months
at 4 ◦ C (mean: 0.0418 ± 0.0016). At 40 ◦ C, the rate of aggregation
increased with the temperature, and a linear increase in optical
density (r2 = 0.95) was observed. After six months at this temper-
ature, an increase in optical density was found, but remained low
(0.0614 ± 0.0001).
3.2. Tertiary structure analysis
No modification or shift of spectrum was observed during the
six-month storage at 4 ◦ C for the peaks corresponding to pheny-
lalanine (260 nm) and tyrosine/tryptophan (285 nm). However, as
showed in Fig. 1, there was a significant structural change after
 M. Paul et al. / International Journal of Pharmaceutics 436 (2012) 282–290 285

Table 1
Mean hydrodynamic (nm) diameter of diluted RTX (1 mg/mL) after different storage conditions.

Storage (days) Main peak 2nd Peak 3th Peak PdIa

◦ ◦
4 C, 25 C and up to 6 months 11.18 ± 0.24 − − 0.07 ± 0.030
40 ◦ C 1 month
40 ◦ C 3 months 11.6 ± 0.32 90.04 ± 11.6 600.7 ± 35.7 0.38 ± 0.038
40 ◦ C 6 months 11.32 ± 0.55 72.6 ± 19.5 527.1 ± 148 0.49 ± 0.039
a
Polydispersity index.

1,2

0,8

%fu
0,6
Tm = 71.7 ± 0.1 Tm = 72.5 ± 0.3 °C

0,4

0,2

Fig. 1. Second derivative UV spectra of samples obtained immediately after recon-
stitution (red line) or after storage at 40 ◦ C during 1 month (blue line) and 6 months
(black line).The concentration of samples was 0.4 mg/ml. The largest peak position
shifts were observed in the minimum at 299 nm and in 3 maxima at 301, 296 nm and
269 nm. One nm shift was observed for one minimum and 3 maxima as indicated
by narrows. (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of the article.)
six months at 40 ◦ C. Weak red shifts (1 nm) were observed in the
maximum at 299 nm and in 3 minima at 301, 296 nm and 269 nm,
respectively, suggesting an alteration of the tertiary structure, and
demonstrating that the method is a good indicator of changes in
stability.
3.3. Determination of hydrodynamic diameters
The results are reported in Table 1. The initial mean hydrody-
namic diameter of RTX was 11.18 ± 0.24 nm, as expected for the
monomeric form of a mAb of about 150 kDa (Matheus et al., 2006).
There was no significant change in this value in any of the storage
conditions up to 180 days at 4 ◦ C or one month at 40 ◦ C. Moreover,
a low polydispersity index (PdI < 0.1) was found. However, the size
distribution by intensity showed two additional peaks at approxi-
mately 100 nm and 600 nm for the storage under stress conditions
after 90 days. Moreover, the cumulative distribution showed poly-
dispersity (PdI = 0.49).
3.4. Thermal denaturation
Fig. 2 presents the evolution of aggregated fractions of RTX
at different temperatures. A typical sigmoidal plot was obtained
0
40 50 60 70 80 90
Temperature °C
Fig. 2. Thermal denaturation curves of Rtx (1 mg/ml). The evolution of aggregated
fractions of Rtx in function of temperature was followed by the determination of
mean diameter using dynamic light scattering. No difference was observed between
curves at D0 and D180 at 4 ◦ C. The Tm was not different after 6 months of storage
at 4 ◦ C. After 6 months at 40 ◦ C, the Tm value was lesser than this obtained after
reconstitution. Each curve represents the mean ± SD of 3 separate experiments. To
(䊉), T6 months (). At 40 ◦ C: T6 months (). In all cases, a typical sigmoidal plot was
obtained (r2 = 0.99).
in all cases (r2 = 0.99). The Tagg values determined from this
curve at D0 and D180 at 4 ◦ C were not different (72.5 ± 0.3 ◦ C
vs. 72.7 ± 0.7 ◦ C) (Table 2). Nevertheless, at 40 ◦ C, a decrease was
observed (71.7 ± 0.1 ◦ C vs. 72.5 ± 0.3 ◦ C; p > 0.001 vs. 4 ◦ C). The H
observed were always endothermic, in agreement with published
data for a thermal denaturation process. Storage at 40 ◦ C induced
a decrease in enthalpy and entropy parameters of the unfolding
process as compared to storage at 4 ◦ C (Table 2). Moreover, this
decrease was also observed with the mT parameter.
3.5. Secondary structure analysis
No modification of the FT-IR spectra was observed after six
months at 4 ◦ C. The spectra of D0 and D180 samples were super-
imposable, and no shift was noted (considering both near and
far IR). The similarity coefficient was close to one (0.968 ± 0.016
vs 0.969 ±0.005 at D0 and after 6 months at 4 ◦ C, respectively).
In Table 3, the percentages of the different secondary structures
are presented, calculated from the amide I bands. The initial

Table 2
Tm values and thermodynamic parameters of RTX calculated for different storage conditions.

Different storage conditions Tm (◦ C) mT (cal mol−1 K) S (cal mol−1 K−1 ) H (kcal mol−1 )

D0 72.5 ± 0.3 1613 ± 42.7 1433.0 ± 78.6 495.0 ± 26.9

D6 months 4 ◦ C 72.7 ± 0.7 1910 ± 17.4 1262.0 ± 91.3 425.0 ± 41.7
D3 months 40 ◦ C 71.9 ± 0.4 456 ± 13.8 1195.0 ± 35.3 415.0 ± 12.5
D6 months 40 ◦ C 71.7 ± 0.1 266 ± 14.5 1124.0 ± 48.6 387.5 ± 36.8
286 M. Paul et al. / International Journal of Pharmaceutics 436 (2012) 282–290

Table 3
FT-IR deconvoluted amide I band frequencies, assignments and percentage for Rituximab (16 mg/mL) stored in different temperatures in Nacl 0.9%.

RTX solution Conditions Frequencies (cm−1 ) Assignments % spectra band assignment 2nd derivative

Just after dilution ␤ Structures 49.1 ± 2.1

1614, 1621, 1628, Intermolecular ␤-sheet 8.9 ± 1.8
1644, 1689 Intramolecular ␤-sheet 24.6 ± 3.1
1633 ␤-sheet and extended strand 15.6 ± 1.7
1651 Random 20.3 ± 1.8
1659, 1668, 1674, 1681 ␤- turn 30.6 ± 2.2
1656 ␣-helix 0

After 3 months at 4 ◦ C ␤ Structures 51.7 ± 2.8

1614, 1621, 1628 Intermolecular ␤-sheet 7.8 ± 0.7
1644, 1689 Intramolecular ␤-sheet 27.7 ± 4.6
1633 ␤-sheet and extended strand 16.2 ± 0.7
1651 Random 20.9 ± 2.7
1659, 1668, 1674, 1681 ␤- turn 27.4 ± 1.9
1656 ␣-helix 0

After 6 months at 4 ◦ C ␤ Structures 51.1 ± 1.7

1614, 1621, 1628 Intermolecular ␤-sheet 8.0 ± 0.4
1644, 1689 Intramolecular ␤-sheet 29.0 ± 4.4
1633 ␤-sheet and extended strand 14.1 ± 2.0
1651 Random 18.5 ± 0.4
1659, 1668, 1674, 1681 ␤- turn 30.4 ± 7.3
1656 ␣-helix 0

After 6 months at 40 ◦ C ␤ Structures 47.6 ± 2.3

1614, 1621, 1628 Intermolecular ␤-sheet 8.2 ± 5.1
1644, 1689 Intramolecular ␤-sheet 22.9 ± 2.5
1633 ␤-sheet and extended strand 16.5 ± 1.4
1651 Random 24.2 ± 1.8
1659, 1668, 1674, 1681 ␤- turn 28.2 ± 0.6
1656 ␣-helix 0

percent of ␤-sheet was approximately 50%. However, although
there was no change in native ␤-sheet content in temperature-
induced aggregates (47.6% ± 2.3 vs. 49.1% ± 2.1; NS), minor
changes were observed for other ␤-structures: random structure
(24.2% ± 1.8 vs. 20.3% ± 1.8); and ␤-sheet and extended strand
(15.6% ± 1.7 vs. 16.5% ± 1.4; NS), which were non-significant, prob-
ably because of the low number of samples tested (n = 3). However,
far IR shifts were observed as storage duration and tempera-
ture increased (Fig. 3), explaining the similarity coefficient found
(0.7624 vs. 0.9695). These results indicate that storage of diluted
RTX solutions at 40 ◦ C, but not at 4 ◦ C, induces modifications in RTX
secondary structure.
rt = 1.4 ± 0.05 min was found in all chromatograms corresponding
to the buffer. In the optimal storage conditions (4 ◦ C), no signifi-
cant change in chromatogram profiles was detected, even after six
months. There was no significant difference between the AUC of
the reference sample and the other samples stored at 4 ◦ C up for six
months, while a decrease in AUC was observed at 40 ◦ C from three
months and reaching 45% at six months. Two new peaks were found
just before the main peak after six months at 40 ◦ C. The retention
times were 2.9 min and 3.3 min, respectively (Fig. 4).
3.7. Size-exclusion chromatography (SEC)

3.6. Cation exchange chromatography (CEX)
A major peak with a retention time (rt) = 4.27 ± 0.02 min was
observed for the reference sample. A minor second peak with a
Only one peak for the RTX reference sample was found. The
retention time of the reference sample was 19.02 ± 0.01 min,
and the mean AUC was 79.47 ± 3.93 mAU min. The molec-
ular weight found was 147.5 ± 1.0 kDa, as expected (Vermeer
and Norde, 2000a). A second peak, at 22.7 min, was observed

Fig. 3. Second-derivative IR spectra of RTX in the 1700–1600 cm−1 region (amide I bands) obtained immediately after reconstitution (blue line) or after storage at 40 ◦ C
during 6 months (black line). After 6 months of storage, modifications are observed. Changes were observed for ␤-structures (random structure and ␤-sheet and extended
strand). Far IR shifts were also observed. The samples were concentrated to 16 mg/mL using 10 kDa Centricon concentration devices (Millipore).
 M. Paul et al. / International Journal of Pharmaceutics 436 (2012) 282–290 287

Fig. 4. Cation-exchange chromatographic profile of RTX (1 mg/ml) using a WCX-10

column at pH 6.0 eluted with a linear salt gradient from 0 to 50% of 1M NaCl and
detection at 280 nm. Decrease in AUC was observed at 40 ◦ C from three months and
reaching 45% at six months. Changes of shape of main peak were also observed. Two
new peaks were found just before the main peak after six months at 40 ◦ C (inset).
for all conditions corresponding to the buffer. Regardless of
the conditions of storage, the retention time and AUC did not
change up to three months. No significant change in chro-
matogram profiles was observed for RTX samples stored for
six months at 4 ◦ C. Nevertheless, after six months at 40 ◦ C, a
weak fragmentation was found (Fig. 5) corresponding to an addi-
tional peak eluting after the original form (rt = 20.8 min). The
Fig. 5. Elution profile of RTX (1 mg/ml) chromatography using a TSK-GEL G4000
SWXL column with detection at 280 nm. Samples were centrifuged before injection.
Stressed sample of diluted RTx (1 mg/ml) stored at 40 ◦ C for 6 months exhibiting an
additional peak at about 22 KDa (light chain) as result of structural breakage.
molecular weight of this fragment was estimated to be
22.1 ± 1.2 kDa. Moreover, after six months at 40 ◦ C, a decrease in
AUC was found (−12.8%), while the decrease only reached 5% after
three months at 40 ◦ C. Finally, no additional peak of higher molec-
ular weight was detected.

Fig. 6. Peptide map of RTX (1 mg/ml). Samples were digested by trypsin and the generated peptides were analyzed by HPLC on a C18 column in gradient mode at 280 nm. A
difference appeared between samples stored at 4 ◦ C (black line) and at 40 ◦ C (blue line), indicating a chemical degradation of the mAb primary structure. (For interpretation
of the references to color in this figure legend, the reader is referred to the web version of the article.)
288 M. Paul et al. / International Journal of Pharmaceutics 436 (2012) 282–290

Table 4
Cytotoxicity of RTX stored in different conditions on RAJI cells.

D0 4 ◦C 40 ◦ C

D30 D180 D30 D180

Number of experiments 10 2 5 3 2
IC50 (␮g/mL) 137.2 ± 28.18 134.7 ± 21.6 125.8 ± 36.5 331.7 ± 32.2b 573.9a ± 181.1b
IC25 (␮g/mL) 41.9 ± 8.3 45.6 ± 60.56 35.8 ± 11.9 83.5 ± 8.8b 119 ± 33.9b
R2 0.994 ± 0.035 0.986 ± 0.014 0.993 ± 0.031 0.992 ± 0058b 0.996 ± 0.002b
AUC 25,334 ± 2316 25,785 ± 1284 25,077 ± 2451 17,910 ± 329b 14,301 ± 433.2b
a
By extrapolation.
b
p < 0.01 vs D0.

80 (1 mg/mL in saline) stored at 4 ◦ C in polyolefine bags remained sta-

ble for at least six months. No physical or chemical instability was
found, and the biological activity was fully maintained. No visible
70
aggregation was found by turbidimetry. SEC did not show poly-
merization or fragmentation, and retention time and AUC were
60 identical for the control and the solutions stored for six months at
4 ◦ C. No significant difference was found considering the hydrody-
% of inhibition

50 namic diameter of RTX, in all dilutions and storage conditions, up to

40
30
20
10
0 This gives mAbs a good stability and resistance to moderate ther-
0 100 200 300 400
RTX concentration (µg/ml)
Fig. 7. RTX direct cytotoxic on CD20-expressing cells was determined through
concentration-dependent cytotoxicity curves (concentration of Rtx: 0, 4.5, 9, 22.5,
45, 90, 135, 270 and 450 ␮g/mL) on a human lymphoma cell line (RAJI lymphoma
cells (ATCC number: CCL-86)). Twelve wells were seeded for each concentration
tested. The results were expressed as the percentage of inhibition in comparison
to control (without RTX). The cytotoxic effects of RTX were not different after six
months of storage at 4 ◦ C.
3.8. Peptide mapping
No change in chromatographic profile was observed for samples
stored at 4 ◦ C for six months. At D90, a difference appeared between
samples stored at 4 ◦ C and at 40 ◦ C (Fig. 6), indicating a chemical
degradation of the mAb primary structure.
3.9. Direct cytotoxicity assay
A shown in Table 4 and Fig. 7, the cytotoxic effects
of RTX were not different after six months of storage at
4 ◦ C. Indeed, the IC50 and AUC were not different (137.2 ±
28.18 ␮g/mL vs. 125.8 ± 36.5 ␮g/mL and 25,334 ± 2316 vs.
25,077 ± 2451 ␮g/mL/min, respectively). In contrast, storage
at 40 ◦ C induced a significant decrease in RTX cytotoxic proper-
ties (IC50 × 4.2) associated with an important decrease in AUC
(45%), thus demonstrating a severe alteration of the mAb biologic
activity.
4. Discussion
The analysis of diluted RTX stability using several different
analytical and biological methods demonstrated that diluted RTX
three months. No additional peak or decrease in the AUC was found
by CEX. The thermal aggregation curves and their derived ther-
modynamic parameters were unchanged, suggesting the absence
of intra- or intermolecular interactions, and a conserved struc-
tural integrity without appreciable destabilization. The primary,
secondary and tertiary structures of the protein were not modi-
fied as demonstrated by peptide mapping, and second derivative
IR and UV spectroscopy. Finally, the direct cytotoxic effect of RTX
was fully maintained.
These results are not surprising, considering the presence of a
significant number of disulfide bonds, intimate domain–domain
interactions and the high content in ␤-sheets (Wang et al., 2007).
500
mal stress, as compared to other proteins and revealed by Tm or Tagg
values higher than in other proteins (range of 70–80 ◦ C vs. range of
40–65 ◦ C for globular protein) (Wang, 1999). Therefore, it is surpris-
ing that the manufacturer recommends the use of RTX within 24 h
after dilution. This recommendation could be only based on bacte-
riological criteria, because it is not supported by physicochemical
and biological studies of instability.
Several previous studies demonstrated the good stability of
several mAbs. Liu et al. showed that a high temperature was
necessary to observe clear physicochemical instability (six months
at 40 ◦ C) (Liu et al., 2006). Usami et al. found that storage at 37 ◦ C for
14 days did not alter the tested mAb (Usami et al., 1996). Cordoba
showed that four humanized IgG1 were stable for one month at 5 ◦ C
(Cordoba et al., 2005). Finally, Bakri indicated a six-month stabil-
ity at 4 ◦ C for diluted bevacizumab (Bakri et al., 2006). However,
this study was only based on immunoassay methods measur-
ing bevacizumab concentration, and very recently, it has been
suggested that these analytical methods are insufficient to prove
the stability of these complex molecules (Sreedhara et al., 2011).
Indeed, the combination of several orthogonal methods, each hav-
ing its own characteristic measuring principle, for example, by size,
quantification or structure, should be performed to study the sta-
bility of biological products (Mahler et al., 2009). Therefore, Pabari
demonstrated that trastuzumab in solution was resistant to vari-
ous processing stresses (sonication, freeze–thawing, lyophilization
and spray drying) using different orthogonal methods (physico-
chemical and biological studies) (Pabari et al., 2011). The use of
such orthogonal methods is also suggested in the current European
Medicines Agency (EMA) draft guideline on “Production and quality
control of monoclonal antibodies and related substances” (Mahler
et al., 2009) and also by the recent consensus conference on sta-
bility studies for anticancer drugs (Bardin et al., 2011). According
to these recommendations, all methods employed in this study,
 M. Paul et al. / International Journal of Pharmaceutics 436 (2012) 282–290
widely used for protein analysis, are appropriate to determine a
beyond-use date.
To obtain information on the most susceptible degradation
pathways and to confirm that the analytical methods were good
indicators of stability, we exposed RTX under an elevated temper-
ature stress condition. We chosen a temperature of 40 ◦ C because
it seemed to be a better threshold for studying stability than higher
temperatures (Duddu and Dal Monte, 1997).
Physical instability, which is usually the first response to stress
of high molecular weight molecules such as mAbs, was observed
only in stress conditions and after three or six months. Optical den-
sity, which is a good a marker of visible aggregation, remained low
(0.0614 ± 0.0001 vs. 0.0418 ± 0.0016) even after 40 ◦ C for 6 months,
confirming that aggregation is not the major degradation pathway
for IgG mAbs after thermal stress (Liu et al., 2006), as opposed
to what is observed under mechanical stress (Lahlou et al., 2009;
Sreedhara et al., 2011). The results of the DLS analysis, which is
very sensitive in detecting submicron aggregates (also described
as “subvisible” or “soluble” aggregates), revealed two subpopu-
lations with a 9-fold and 60-fold higher hydrodynamic diameter
(90.0 ± 11.6 nm and 600.7 ± 35.7 nm), as compared to the reference
sample (11.18 ± 0.24 nm) when diluted RTX was stored for 90 days
at 40 ◦ C. These results suggest the presence of subvisible aggregates
that were not detected by turbidimetry, in accordance with pre-
vious studies of protein aggregation under thermal (Vermeer and
Norde, 2000b; Hawe et al., 2009) and mechanical stress (Mahler
et al., 2005; Lahlou et al., 2009). SEC also revealed that there was
no subvisible aggregation after six months at 4 ◦ C. The absence of
aggregation after six-month storage at 4 ◦ C is very important, con-
sidering the possible induction of allergic toxicity due to particles or
aggregates, even at a very low level, which can be initially present
or formed during biological drug handling (Sharma, 2007a,b).
A weak fragmentation of the RTX molecule was demonstrated
by SEC (Fig. 5), but only after six months at 40 ◦ C. SEC revealed
one additional peak downstream of the main peak, corresponding
to a lower molecular weight than IgG as previously described by
Cordoba (Cordoba et al., 2005). The molecular weight of this frag-
ment was estimated at 22.1 ± 1.2 kDa, which is coherent with the
release of light chains, as previously reported by Liu (23.5 kDa) after
6 months at 40 ◦ C (Liu et al., 2006).
Regarding chemical stability after storage at 40 ◦ C, significant
modifications were observed with all methods employed. CEX is
a useful tool to detect subtle protein alterations such as deamida-
tion or oxidation (Moorhouse et al., 1997). These alterations could
modify the primary and secondary structure of RTX. Indeed, two
increasing distinct peaks with a shorter retention time (2.9 and
3.3 min, respectively) than the reference sample (4.27 min) were
observed in samples stored at 40 ◦ C, demonstrating a modification
of the charge of RTX molecules, and the formation of more acidic
derivatives, which can reflect both global charge or local charge
modifications, as shown in Fig. 4. Furthermore, according to the lit-
erature on deamidation and isomerization, a C-terminal position or
succession of Gly, His or Ser, or Asp and Asn residues can influence
the protein degradation rate (Wakankar and Borchardt, 2006). Con-
sidering the primary structure of RTX, both of these conditions are
met, as Gly and Ser succeed to Asp inside the heavy chain, and Asp
is in the C-terminal position inside the light chain. Finally, at pH
5 (pH 6.5 in the RTX formulation), an increase in Asn deamidation
rates was observed (Manning et al., 2010).
Furthermore, peptide mapping, which is a sensitive finger-
print process method including enzymatic and chemical cleavage,
revealed changes in the chromatographic profiles (Fig. 6) with
four additional peaks located within the 70–110-min elution zone.
These additional peaks could be the result of Asn or Gln deami-
dation (Liu et al., 2006), and could induce conformational and/or
289
structural changes, as shown previously (Wan et al., 1999; Harris
et al., 2001).
The modifications of the tertiary structure, induced by an
increase in temperature, is probably due to changes in the degree of
protonation/deprotonation or ionization of aromatic amino acids,
such as tyrosine, tryptophan or phenylalanine. In accordance with
this hypothesis, we observed a 1 nm red shift for three minima
and one maximum, probably due to an electronic differentiation
process, as previously described by Balestrieri et al. (1980).
Considering the secondary structure, we found that the initial
percentage of ␤-sheets is lower (50% vs 65%–70%) and the per-
centage of random coil is higher (20% vs 5%) than those previously
published for IgG by different authors (Dong et al., 1997; Matheus
et al., 2006). This difference could be due to the presence of buffers
and surfactant agents in the commercial solution of rituxumab.
Indeed, the addition of phosphate buffer induces an increase of ran-
dom coil (10%) (Vermeer et al., 1998). The adjunction of surfactant
allows obtaining 50% of ␤ − sheet structure with 20% of random coil
structure (Vermeer and Norde, 2000a) that explains our results.
All RTX modifications observed were due to an increase in tem-
perature, and it is well known that increased temperature decreases
protein stability in thermodynamic terms by favoring unfolded
states and by increasing disorders in the system (Speed et al., 1997;
Wang, 1999). Protein stability results from an equilibrium between
destabilizing and stabilizing forces. Destabilizing forces are mainly
due to a large increase in unfolding entropy (dissipative forces, loss
of conformational entropy of native state), and stabilizing forces
(intramolecular interactions) are mainly due to non-covalent inter-
actions
Different parameters can be studied to estimate the degree of
internal or external stabilization, such as the changes in unfolding
(Tm ) or aggregation (Tagg ) temperature. As shown in Table 2, the Tagg
values obtained in our study were as high as those found in the lit-
erature (Tm IgG: 73.6 ◦ C ± 0.4 (Liu et al., 2009), 72.6 ◦ C (Vermeer and
Norde, 2000b), 74 ◦ C (Wang, 1999; Matheus et al., 2006), indicat-
ing a high resistance to temperature (the higher the Tm , the greater
the thermal resistance of a protein). The storage of RTX during a
long period of time and at a high temperature (40 ◦ C) led to a slight
decrease in Tagg (−0.8 ◦ C), suggesting a low but significant alter-
ation of the protein integrity, because when the integrity is strongly
altered (breaking of disulfide bonds), the decrease of this parameter
is more important (>1 ◦ C) (Wang, 1999).
We observed that all thermodynamic parameters were posi-
tive, thus corresponding to an endothermic process as described
for other proteins. Because H and G are positive, aggregation
is not possible without an increase in temperature. As shown in
Table 2, the initial value of H is high (about 500 kcal mol−1 ) and
close to values found by Matheus (578 kcal mol−1 ) or Vermeer
(556 kcal mol−1 ) (Vermeer and Norde, 2000b; Matheus et al., 2006).
After a long storage period at 40 ◦ C, a major decrease in all thermo-
dynamic parameters (S, H) is noticed. The extended stability of
diluted RTX stored at 4 ◦ C was finally ascertained by verifying that
its cytotoxic properties were maintained. Indeed, a major part of
the biological activity of RTX is related to its ability to bind CD20-
expressing cells. After storage of diluted RTX for six months at 4 ◦ C,
using a human lymphoma cell line, we demonstrated that dose-
dependent cytotoxicity curves were fully superimposable with no
difference between DL50 values. In the opposite, RTX maintained in
stressed condition (30 days at 40 ◦ C) have lost about 50% of its bio-
logical activity. Interestingly, this dramatic lost of biological activity
was observed even analytical method indicated indeed detectable
alterations but which seemed limited. These results suggest that if
several analytical methods are mandatory to assess long-term sta-
bility of mAbs, they should be completed by an estimation of their
biological activity.
290 M. Paul et al. / International Journal of Pharmaceutics 436 (2012) 282–290
5. Conclusions
Our results demonstrate that diluted RTX (1 mg/mL in saline)
remains stable for at least six months when stored in polyolefin
bags at 4 ◦ C. Indeed, no modification of its chemical, physical and
structural characteristics and no aggregation were observed. This
conclusion is supported by the results of several orthogonal meth-
ods, as recommended in the stability assessment of biological
products. Moreover, the pharmacological properties of RTX were
fully conserved. These results indicate that the time frame for the
practical use of diluted RTX can be safely extended, allowing, for
example, early reconstitution of workload optimization or safe
storage of a patient’s bags for several days if the RTX cannot be
immediately administered. Our results also support the possibility
of preparing standardized batches of “ready-to-use” diluted RTX
under good manufacturing procedures to ensure maximal secu-
rity in terms of sterile conditions and quality controls. Finally, this
extended in-use stability can help pharmacists to optimize hospital
expenses due to this costly drug.
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