Impact of Buffer, Protein Concentration and Sucrose Addition on the Aggregation and Particle Formation during Freezing and Thawing
Impact of Buffer, Protein Concentration and Sucrose Addition on the Aggregation and Particle Formation during Freezing and Thawing
Astrid Hauptmann 1 2 3 5
Katja Podgoršek 1 2 3 5
Drago Kuzman 1 2 3 5
Stanko Srčič 1 2 3 5
Georg Hoelzl 1 2 3 5
Thomas Loerting 1 2 3 5
0 Freezing temperature T
1 Sandoz GmbH , 6336 Langkampfen , Austria
2 Faculty of Pharmacy, University of Ljubljana , 1000 Ljubljana , Slovenia
3 Lek Mengeš , 1234 Mengeš , Slovenia
4 Thomas Loerting
5 ABBREVIATIONS DLS Dynamic light scattering DSC Differential scanning calorimetry FT Freeze and thaw mAb Monoclonal antibody MFI Micro flow imaging OCM Optical cryomicroscopy SEC Size exclusion chromatography T
Purpose This study addresses the effect of freezing and thawing on a therapeutic monoclonal antibody (mAb) solution and the corresponding buffer formulation. Particle formation, crystallization behaviour, morphology changes and cryoconcentration effects were studied after varying the freezing and thawing rates, buffer formulation and protein concentration. The impact of undergoing multiple freeze/thaw (FT)cycles at controlled and uncontrolled temperature rates on mAb solutions was investigated in terms of particle formation. Methods Physicochemical characteristics were analysed by Differential Scanning Calorimetry whereas morphology changes are visualized by cryomicroscopy measurements. Micro Flow Imaging, Archimedes and Dynamic Light Scattering were used to investigate particle formation. Results Data retrieved in the present study emphasizes the damage caused by multiple FT-cyles and the need for sucrose as a cryoprotectant p re venting col dcrystallization specifically at high protein concentrations. Low protein concentrations cause an increase of micron particle formation. Low freezing rates lead to a decreased particle number with increased particle diameter. Conclusion The overall goal of this research is to gain a better understanding of the freezing and thawing behaviour of mAb solutions with the ultimate aim to optimize this process step by reducing the unwanted particle formation, which also includes protein aggregates.
buffer and mAb formulation; cryomicroscopy; freezing; particle formation; thawing
1 Institute of Physical Chemistry, University of Innsbruck, Innrain 52c
6020 Innsbruck, Austria
The development and optimization of freezing and
thawing (FT) processes of therapeutic proteins as
pharmaceutical products are among the challenges encountered
by the pharmaceutical industry. Even though the process
is used routinely in industry, it comes with some
disadvantages. Compared with room temperature storage the
FTprocess is time-consuming and costly and may result in
quality loss of the protein (
). The benefits outweighing
these disadvantages include foam prevention and
mechanical stress reduction during transportation, decrease of
bioburden and microbial growth during storage and
enhancement of stability of the product by slowing down
any kind of degradation reaction rates that could lead to
). Like all other therapeutic proteins,
monoclonal antibodies (mAb) may undergo structural changes
during FT processes which lead to protein aggregation,
misfolding/unfolding, loss of biological activity or
enh a n c e d i m m u n e r e s p o n s e i n p a t i e n t s (
5 – 7
) . T h e
aggregation process is generally poorly understood (
may have several causes, such as cryoconcentration (
contact with interfaces (
), cold denaturation
), and many others (
In this work the focus lies on the systematic search for a
good buffer formulation and freeze-thaw protocol for an
inhouse chimeric monoclonal antibody to reduce aggregation,
which is a common goal of many pharmaceutical industries
). To this end, the influence of protein concentration as well
as addition of sucrose as stabilizer was scrutinized.
Furthermore, the impact of freezing and thawing rates and
the impact of several freeze/thaw cycles on particle number
were studied using a range of methods detailed below.
It is, thus, one of the goals of this study to minimize
formation of aggregates, which we synonymously also call particles.
Here, »particles« is an umbrella term for all kinds of
agglomerates regardless whether the particles are originating from the
protein monomer or from other sources. Aggregates may be
categorized according to size, type of intermolecular bonds,
reversibility, morphology or hydrophobicity (
classified according to their size protein aggregates are subdivided
into visible (>100 μm), micron (1–100 μm), submicron (100–
1000 nm), and nanometer particles (<100 nm) (
aggregation and/or denaturation of the protein, FT-processes
can also lead to an accumulation of leachables (
formation of sub-visible particles (
). Sub-visible protein
particles (typically 0.1–10μm) are too big to be analyzed by
size-exclusion chromatrography (SEC) but too small to be
visible by an unaided eye (7). These particles could be the
potentially most immunogenic class of protein aggregates
and may act as nuclei (
) and cause formation of larger
particles over time (
). In order to detect and assign
aggregates/particles to the categories mentioned above we
have employed Micro Flow Imaging (MFI) and Dynamic
Light Scattering (DLS) (
). MFI is used for detecting
particles in size ranges from 2 to 300 μm and DLS to detect and
characterize soluble aggregates on a length scale of ca. 1–
800 nm (
In order to avoid aggregation in the frozen solution the
storage temperature should be chosen low enough, so that
the solution as a whole has turned into a solid. This is the
case below the glass transition temperature of the
freezeconcentrated solution (Tg’) (
). Since Tg’ of a protein
solution is determined by the concentration and nature of
the cosolutes, buffer composition, pH and ionic strength it
is necessary to establish an optimal formulation (
Optical cryomicroscopy (OCM) and differential scanning
calorimetry (DSC) are employed here to determine
freezing temperatures, supercooling or Tg’ from the latter and
an analysis of the distribution of solid and liquid upon
freezing and thawing from the former. In other words,
the effects of cryoconcentration are studied using the
combination of a thermal and optical image method. It is
useful to distinguish between micro- and
macrocryoconcentration in this context (37).
Microscopic scale cryoconcentration is based on the
unavoidable dehydration of the liquid phase when water
molecules form ice crystals. Solutes are trapped in the interdentritic
space and increasingly freeze-concentrate until they reach the
maximally freeze-concentrated solution (MFCS) (
). In a
state diagram, such as the one for sucrose-water depicted in
Fig. 1, the MFCS is given by the intersection of the melting
(Tm) and glass transition (Tg) lines at the point denoted
Tg,max’. This is the case at roughly 80 wt% in Fig. 1 and
implies that freezing takes place at the melting line. In real
experiments, however, supercooling plays an important role,
and the solutions might remain in the liquid state down to the
homogeneous nucleation temperature (Thom in Fig. 1). In this
case, the dehydration incurred upon ice freezing may result in
a freeze-concentrated solution of lower Tg, which is less
concentrated than the MFCS. In the example of sucrose in Fig. 1
this may result in vitrified solutions of 64 wt% sucrose, as
defined by the intersection of Thom and Tg at Tg,min’.
Macroscopic scale cryoconcentration on the other hand
happens when solutes or protein are progressively pushed into
a certain direction in the bulk due to the growing ice crystal
front of the water and the exclusion from the solid-liquid
). Macroscopic scale cryoconcentration is very hard
to control since it depends on a large number of parameters,
including the size of the sample, the temperature gradients in
t h e s a m p l e , e t c . W h e n b o t h m i c r o - a n d m a c r o
cryoconcentration occur two distinct freeze-concentrated
solutions, FCS1 and FCS2, may be observed (
Fig. 1 State diagram of sucrose-water, detailing melting temperature Tm,
homogeneous nucleation temperature Thom, glass transition temperature Tg
and crystallization temperature Tx. The freeze-concentrated solution will
show a glass transition temperature anywhere between Tg,min’ and Tg,max’.
Data taken from reference (
The cooling rate has a large influence on the degree of
supercooling, and consequently the concentration of FCS1 and
FCS2 as well as the number of ice nuclei. These factors together
in turn affect the formation of aggregates. The slower the
freezing the more freeze-concentration on the macroscopic scale plays
a role (
), which may lead to partial unfolding and loss of
protein activity (
). Fast freezing may lead to formation of a very
large number of small ice dendrites, therebye increasing the size
of the ice-liquid interface (
) and creating a large number of
interdendritic spaces (
). Also this may be unfavorable for the
), but was also suggested to favor protein stability
due to more uniform protein distribution (
Slow thawing may result in cold-crystallization, i.e., growth
of small ice nuclei to small ice crystals from devitrified
freezeconcentrated solution above Tg’ (
). Consequently, the
optimal freezing-thawing rate cannot be stated in general, but may
differ from solution to solution. Usually slow freezing and faster
thawing rates are preferred (
). In order to minimize the
damaging effects cryoprotectants/cryostabilizers maybe helpful
). This strategy is often used for protein solutions
). Usually formulation additives such as sugars,
aminoacids, polyvalent alcohols, oligosaccharides or polymers
are used in order to stabilize the protein conformation and to
prevent its adsorption on the surface of ice crystals. It is, thus, of
importance to optimize the formulation, too. Sucrose and
trehalose are popular examples for such additives in protein
solutions. In this context, it is important to note that these additives
should not crystallize themselves but turn into the glassy state.
Furthermore, it is of importance to control the solution pH – at
low pH sucrose in solution can cause glycation (non-enzymatic
glycosylation) of the monoclonal antibody (
). This is
usually done by employing buffers, such as phosphate or citrate,
which also need to stay amorphous upon freezing and thawing
(47). For this reason we here study the freezing of a buffer
solution containing a range of protein concentrations with or
without the addition of sucrose as cryoprotectant.
MATERIAL AND METHODS
In this study we are focusing on the aggregate formation of a
monoclonal antibody (mAb) formulation. Protein solutions in
the concentration range 1–30 mg/ml were embedded in a
25mM sodium citrate buffer (pH 6.5) with or without addition
of 125mM sucrose. The following studies were done:
Freezing Box and Celsius-S3 Studies
In order to evaluate the role of freezing/thawing rate and the
number of freeze cycles on aggregate formation samples with a
protein content of 1 mg/ml were placed in a freezing box at
different positions between vials filled with water (Fig. 2). The
freezing box was placed in a -80°C freezer. Temperatures
recorded during freezing have shown the typical shape,
including a linear cooling ramp in the liquid state, a freezing plateau
and another linear cooling ramp in the solid state. For samples
placed in the middle of the box the liquids were cooled at
1.1 K/min, and the freezing plateau of constant temperature
lasted for 9.2 min. For samples placed at the corner, the cooling
rate was found to be higher (3.9 K/min), and the freezing
plateau only lasted for 2.4 min. A supercooling of −4.7°C
was observed. After having cooled to below Tg,min’ the samples
were thawed by bringing them to room temperature again.
This procedure was repeated up to five times.
In another set of experiments the concentration of mAb in the
formulation was varied from 1 mg/ml to 30 mg/ml, with or
without the addition of sucrose. Samples were frozen and thawed
in the Celsius®–S3 Benchtop System (Sartorius, Germany)
(simulating the freezing protocol of our production scale). The system
is temperature controlled by a cooling liquid (Syltherm
HF®HTF) which flows circularly around the cooling chamber.
Temperatures were controlled using the computer program
Cryopilot and 10 thermocouples (Pt 100). The sample holder,
originally made to freeze and thaw plastic Celsius-Paks, was
custom made for placing 1 ml, 1.5 ml and 2 ml Nunc CryoTubes or
4–5 ml PETG vials. For particle characterization the sample was
homogenized by turning the vial 10 times carefully by hand
followed by letting the sample settle for 1 h at ambient
temperature. After turning the sample vials for 10 times again the
samples were aliquoted and measured by using MFI and DLS.
Differential Scanning Calorimetry (DSC)
DSC measurements were conducted on a DSC 8000
(PerkinElmer, USA). Hereby, around 30 μl of sample was loaded into
an Al crucible, hermetically sealed and transferred to the device
at ambient temperature. An empty aluminium pan was used as
a reference. Starting from 10°C the sample was cooled to
−90°C, equilibrated for 5–10 min and re-heated at rates of
1°C/min, 2°C/min, 5°C/min, 10°C/min, 20°C/min and
40°C/min. For the calibration at subzero temperatures the
recommended transitions (
) in cyclopentane, cyclohexane,
indium, and adamantane were used in order to ensure correct
tansformation temperatures with an accuracy of ±1°C. An
accuracy of ±0.1 J/mol K in latent heat and change in heat
capacity can be achieved due to an calibration using sapphire.
Optical Cryomicroscopy (OCM):
Images of frozen buffer solution with or without sucrose were
t a k e n b y a n o p t i c a l m i c r o s c o p e B X - 5 1 ( O l y m p u s
Corporation, Japan) in combination with a temperature
controlled cryostage LTS420 (Linkam Scientific Instruments,
UK). With the Linkam-cryostage subzero temperatures down
to −196°C can be reached by using liquid nitrogen as cooling
medium. A drop of sample (approx. 0.5 μl) was placed on the
object plate and either directly positioned into the
cryochamber without covering, or firstly enclosed by a cover glass
slip before analyzing in order to prevent evaporation. Loaded
at ambient temperatures, samples were next frozen to −80°C
at rates of 1°C/min, 2°C/min, 5°C/min, 10°C/min, 20°C/
min, 30°C/min and 40°C/min and analyzed isothermically
by using standard transmitted light with a crossed-polarized
filter. Crossed-polarized light can be used for visualization of
structural changes, crystal sizes and ortientation in the droplet.
Morphology changes in the frozen form were determined by
calibrating an ULWD 50× objective (Olympus Corporation,
Japan) and using the Linksys 32 software. The sample droplet
had to be analyzed as a thin layer, which could be achieved by
using a top glass positioned on the sample droplet while
measuring. The downside of that method had been the
accumulation of ice crystals formed onto the top glass especially at
lower rates which originated from the humid environment of
the cryo chamber and are a product of condensation.
Micro-Flow Imaging (MFI)
MFI System MFI 5100 (ProteinSimple, Canada) was used to
capture particle images as the sample passes continously
through a 400 μm flow cell in front of a CCD camera.
Micron sized and visible particles and protein aggregates (2–
300 μm) can be depicted in thousands of frames per minute
and extracted by the MFI View System Software where they
are categorized. The Software provides specific information
about the properties like size, number (counts/ml) and
morphology of the particles (
). 5% PCC (Thermo Fischer
Scientific, USA) solution, a mixture of anionic and nonionic
surfactants, and milli-Q-water was flushed through the MFI
device before and after each measurement in order to keep
the system clean and retain a clean base line. 1.5 ml of
undiluted protein solution were injected into the flow cell (0.2 ml
for pre-run/purge volume and 0.615 ml for analysis) and
analysed using a flow rate of 0.22 ml/min.
Archimedes (Malvern, United Kingdom) was equipped with
HiQ microsensor (Malvern) and was used for particle
characterization in the size range from 200 nm to 5 μm. The system was
controlled by the Archimedes Version 1.20 Software. Before
every analysis blanks (UPW/ milli-Q-water) were measured to
ensure a clean system. This is defined by the blank showing less
than 20 particles after 10 min of analysis. Before sample
measurement the system automatically determines the limit of
detection (LOD) which can be subsequently changed for results
comparison and processing. Each sample was loaded for 30 s
and was then analysed (100 nl of sample solution). Between each
sample measurement the system was flushed with milli-Q-water.
Dynamic Light Scattering (DLS)
A Zetasizer APS (Malvern, United Kingdom) supported by the
Zetasizer Software DTS software was used to measure the size
and distribution of nanometer and submicron particles. The
device automatically measures liquid samples in microtiter
plates. After thawing each sample (100 μl) was analysed five
times at ambient temperatures and afterwards discarded. In
addition to protein samples the standard of latex spheres size
60 nm and 200 nm (Duke standards™, Thermo Scientific) was
also measured before and after analysis of samples to ensure
correct operation. The plate filled with samples was protected
with aluminium foil during the analysis.
Particle Numbers and Sizes
First, we show the influence of the number of F/T cycles on
the number (Fig. 3) and circular equivalent diameter (Fig. 4) of
particles forming in 1 mg/ml protein solution without sucrose
in 25 mM sodium citrate buffer. MFI data (Fig. 3) show the
progression of particle numbers with F/T cycles. Each
additional F/T-cycle leads to an increase of particle number and
hence to aggregate formation in the sample. After the fifth
cycle the number of particles is half in the samples positioned
in the middle of the box vs. the edges (Fig. 3), but about 15%
bigger in terms of diameter (see Fig. 4). That is, especially
samples placed at the edges of the freezing box are affected
mostly by the repeated F/T cycles. This clearly demonstrates
that temperature gradients play an important role for the
formation of particles. Smaller temperature gradients found
in the middle of the box are favorable to avoid high particle
numbers, but will lead to larger particles.
Samples with different protein concentration were
analyzed by Archimedes and MFI (see Fig. 5) and DLS (see
Table I) with and without sucrose being added. Higher
protein concentration results in an increased number of
small particles detected by Archimedes (ranging from
Protein formulation without sucrose
Protein formulation with sucrose
200 nm to 5 μm, Fig. 5a). However, simultaneously it
results in a decreased number of larger particles (micron
and visible) in the size range from 2 μm to 300 μm
detected by MFI (Fig. 5b). Figure 5c compares the influence
of the initial protein concentration directly: higher
concentration increases the number of small particles up to
5 μm, but decreases the number of larger particles.
The influence of the number of FT-cycles is best seen in
Fig. 5a, b, in which the three bars detail the number of
particles before the first, after the first and after the fifth FT-cycle.
Archimedes measurements reveal that the particle numbers
increase after the first FT-cycle significantly, but remain
almost constant between the first and fifth FT-cycle, regardless
of the protein concentration. In contrast, MFI results indicate
a drastic increase of micron/visible particles from the first to
the fifth FT-cycle. That is, the number of larger particles
increases with the number of FT-cycles, but the number of the
small particles remains invariant. Focusing on particle sizes
bigger than 4 μm, the decrease of particle number in each
size range shows a linear trend. The trend line of samples with
30 mg/ml protein concentration has a steeper slope than the
one from samples with 1 mg/ml (see Fig. 5c). Since the
number of particles is plotted logarithmically, the linear behaviour
implies particle numbers decreasing exponentially with size.
The influence of the addition of 125 mM sucrose to the
solution can be seen by comparing the left and right panels in
Fig. 5a–c. On first look sucrose does not seem to have a very
large influence on the number of particles. On closer
inspection it can be noticed that the number of particles is actually
increased for the 1 mg/ml protein solutions, but decreased for
the 30 mg/ml solutions. This is true especially for the small
particles after the first freezing cycle (Fig. 5a). After the fifth
cycle, however, the beneficial effect of sucrose on the small
particles has disappeared again. For the larger particles
addition of sucrose, results in a reduction of about one third (Fig.
5b, 30 mg/ml) even after the fifth cycle.
The polydispersity index (PdI) and the hydrodynamic
diameter 2r as obtained from DLS measurements are listed in
Table I. The PdI increases dramatically for samples with
1 mg/ml protein after the addition of sucrose, but is barely
affected for all other solutions by sucrose. Thus, higher protein
concentration enforces monodispersity. Except for the 1 mg/
ml protein solution the PdI increases slightly after 5 FT-cycles.
At the same time addition of sucrose increases 2r by about
1 nm, compared to the solutions without sucrose. Again the
exception is the 1 mg/ml protein solution, for which only the
PdI is increased massively, whereas 2r remains unaffected by
the addition of sucrose. However, 2r increases systematically
with protein concentration, from 11.2 nm at 1 mg/ml to
17.2 nm at 30 mg/ml (Table I). The hydrodynamic diameter
of the mAb monomer is known to be 11.2 nm. This value was
measured in a protein concentration of 1 mg/ml at ambient
temperatures and also in accordance with the value predicted
by using the Hydropro software (
) on the crystal structure of
an intact IgG mAb (
The influence of cooling rates on the morphology of the frozen
samples was studied on individual droplets of millimeter size
using OCM experiments. OCM images allow for resolution of
freezing-induced morpholgocial changes on the μm-length
scale. Figure 6 shows the changes induced upon cooling buffer
solutions without (top two rows) and with sucrose (bottom two
rows), respectively. Generally, pure buffer without sucrose
shows many features after cooling to −80°C. Channels
reminiscent of leaf-veins have grown throughout the droplet (Fig. 6,
top row) and shade the transparent patches of ice crystals with
darker nuances. These veins are a result of
macrocryoconcentration and contain freeze-concentrated buffer
solution. When crossed-polarized light is applied (Fig. 6, second
row) the orientation of the ice crystals and their edges,
junctions and triple junctions become apparent through different
tones of green. After slow cooling at 1 or 2°C/min relatively
large ice crystal platelets of dimensions 100–300 μm can be
defined and located precisely. The maximally
freezeconcentrated solution (MFCS) is located mainly at the edges
of the few larger crystals. After faster cooling at 30–40°C/min
fading and/or loss of the platelet structures together with the
formation of an extended network of veins that pass through
the droplet is observed (Fig. 6, top rows). That is, the ice
crystals are much smaller, and the MFCS is more widely and less
heterogeneously dispersed for faster cooling rates.
Homogeneity increases even more in sodium-citrate buffer
with 125 mM sucrose, which results in opacity of the droplet
in the frozen state (Fig. 6, bottom two rows). Images taken
after freezing at 1 or 2°C/min show a rather homogenously
colored green surface through crossed polarizers. After
freezing at 20 or 40°C/min star-shape like patterns appear when
using crossed-polarized light. Two such stars are observed
after cooling at 20°C/min, and only a single one after cooling
at 40°C/min. We interpret the centers of these stars as
individual nuclei, from which the ice crystal growth has started.
Veins such as the ones in Fig. 6 (top row) are absent when
sucrose is added. This implies that the FCS is much more
homogeneously distributed. Note that the dark spots that
appear both inside and outside the droplets are small ice crystals
condensed from air on top of the cover glass.
The evolution of these star-shaped features with
temperature in sucrose-citrate solution is detailed in Fig. 7. Visual
inspection using the cryomicroscope shows that indeed the
freezing process starts from a single spot, from a single ice
nucleus, and it is followed by crystal growth directed towards
the outer surface. The onset of the growth is observed at
−26.3°C for 2°C/min cooling rate and at −35.4°C for 40°C/
min (Fig. 7, left). The main difference of the freezing process at
2°C/min (Fig. 7, top row) and the one at 40°C/min (Fig. 7,
bottom row) is that with faster rates the star-shape like crystal
formation remains down to the lowest temperatures −60°C
(Fig. 7, bottom right). When slower rates are applied the edges
fade away (see Fig. 7, top right image) and the droplets appear
as homogenously frozen. This homogenization is related to
the second freezing event pertaining to the FCS dispersed
homogeneously in between the rays of the star shape. This
second freezing is only observed for slow cooling rates (see
DSC data below).
DSC measurements provide complementary thermal
information – just like for the OCM study we have employed
millimeter-sized droplets and cooling rates between 1 and
40°C/min. From the calorigrams shown in Fig. 8 we extract
freezing temperatures (Tf) as well as glass transition
temperatures of freeze-concentrated solutions (Tg’) and list
them in Table II. Figure 8 also shows the thermal effects
incurred upon thawing the frozen sample (right).
All DSC scans recorded upon cooling show at least one
massive exotherm, indicating the freezing of ice from the
solution. In some cases the freeze-concentrated solution (FCS)
experiences a second freezing event, whereas in other cases the
first freezing event is followed by vitrification at Tg’ (Fig. 8). The
second freezing exotherm appears only for cooling rates of 1–
5°C/min in the presence of sucrose (Fig. 8, top left). However,
the second freezing exotherm is never observed without the
addition of sucrose (see Fig. 8, bottom left). The second freezing
event for slow cooling rates in the presence of sucrose is
responsible for the homogenization below −27°C observed in Fig. 7 at
2°C/min. In the DSC experiment the onset for the second
freezing event is found to be between −20°C and −25°C, in
agreement with the observations in the cryomicroscope.
Table II Freezing Temperatures Tf, Glass Transition Temperatures Tg’ and Width of Freezing Peak of 25 mM Sodium Citrate Buffer with and Without Sucrose
Determined by DSC Measurements
Cooling and Heating Rates
Buffer without sucrose
Buffer with sucrose
Tf was obtained from DSC cooling scans, Tg’ from the subsequent heating scan. Width is determined as difference of onset and end point of the freezing transition.
Tf is defined by the onset of the exotherm. For a thermodynamically controlled transition the onset temperature is rate invariant, except for thermal lag of the
instrument at fast rates. By contrast, the offset typically shifts with a change of rate since the width of the transition is governed by kinetics, i.e., the time it takes for
the whole sample to undergo the phase transition
*Tf indicates the range of freezing temperatures obtained from repeated measurements
For the first freezing event, i.e., crystallization of ice from
the solution, Tf onset is seen between −7°C to −19°C for the
pure buffer and between −10°C to −18.3°C for the buffer
with sucrose (see Table II). Both freezing ranges are clearly
below the thermodynamic freezing/melting temperature,
which is revealed from the magnified DSC heating scan in
Fig. 9 (right). From the tangent method on the slowest heating
scan the equilibrium melting temperature Tm is evaluated to
be −1.1°C without sucrose and −3.8°C with sucrose (see
tangent intersection in Fig. 9). The difference between the
two represents the melting point depression incurred because
of the addition of sucrose. The supercooling (i.e., difference
between Tf and Tm) is found to be about 6–18°C without
sucrose and 6–14°C with sucrose, but does not depend much
on the cooling rates used here.
Tg’ was determined using the Btangent^ evaluation method
) on magnified versions of the heating curves of the
thermogram (not shown). The Btangent^ evaluation has a
precision of Tg’ ± 3°C. The DSC heat flow is proportional
to the heating rate employed. Since the glass transition
pertains to the FCS in the veins, i.e., only a very minor fraction of
the whole frozen sample, sensitivity is a key issue. Especially at
small heating rates the sensitivity of our instrument is not high
enough to resolve the glass transition for the
freezeconcentrated buffer itself. Only for the heating scans at 20
and 40°C/min the heat flow signal clearly allows to resolve
the increase in heat capacity associated with the glass
transition at −40°C. The glass transition is easier to detect after
sucrose addition, and so we are able to resolve it even on the
slowest heating scans at 1°C/min. As expected for a transition
that is kinetic in origin, it shifts to higher temperatures with
increasing rates. We observe a shift of about +2°C when going
from 1°C/min to 5°C/min (Table II). The addition of
125 mM sucrose shifts Tg’ from −40°C to −32°C at the rate
of 20°C/min, which is the standard rate typically used for the
determination of glass transitions (
). In the sucrose-water
state diagram (see Fig. 1) the MFCS is located at about
80 wt% sucrose, which shows a Tg’ of about −46°C. Our
Tg’ is higher by about 10°C, which indicates that the MFCS
state is not reached. From the sucrose-water state diagram we
estimate that the vitrified FCS in the veins contains about
70 wt% sucrose.
In addition to the devitrification transition at Tg’ we
also observe a weak transition in the heating scans
between −8° and −6°C (Fig. 9). We interpret these small
effects as exotherms and as cold-crystallization of the
devitrified FCS that takes place in the broad
lowtemperature tail of the melting peak. In other words,
freezing and melting events take place simultaneously at
this stage. The same effects were observed for solutions
containing the protein in addition. DSC measurements of
the mAb formulation seem to be very similar to the DSC
curves of the buffer without sucrose (data not shown).
The only difference noticeable is the broad Tg’ and the
absence of the kink in the heating curves when measuring
mAb solution in buffer. Whereas, when sucrose is added
to the buffer it leads to a shift of freezing temperatures
and broadening of the melting peaks (Fig. 8).
The heating scans depicted in Fig. 9 are complemented
with cryomicroscopy heating measurements shown in Fig.
10 (25 mM buffer without sucrose) and in Fig. 11 (25 mM
buffer with 125 mM sucrose). The images were recorded upon
heating at 2°C/min, 5°C/min and 40°C/min (right after
cooling the solution droplets to −80°C at the same rate).
Inspecting the images closely reveals that the melting events
start at the veins. For instance, at −10°C a lot of liquid
droplets are seen along the veins, whereas there are much less little
droplets in the other regions of the the frozen drop. At −4°C
the molten droplets are much more homogeneously
distributed over the whole sample. That is, as expected from
thermodynamics the melting event of ice crystals starts in the locations
of highest solute concentrations, specifically in the veins
containing the FCS. With the addition of 125 mM sucrose the
first melting event shifts by about 3°C to lower temperature,
consistently seen in the OCM images (Figs. 10 and 11) and the
DSC measurements (Fig. 9).
MFI data of the samples frozen at −80°C and thawn to
room temperature show a correlation between position in
the freezing box and the extent of particle formation. In
the middle of the freezing box vials are freezing and
thawing slower than vials placed at the edge due to the
temperature gradient driven by the passive heat transfer.
Fig. 10 OCM images at the indicated temperatures recorded upon heating at the indicated rates for frozen 25 mM sodium citrate buffer solutions without
This experimental set-up proves that cooling/heating
rates have an impact on the number of nuclei, the ice
crystal size and the amount and size of particles (Figs. 3
and 4). Slow cooling rates lead to fewer but bigger (ice)
), and this in turn results in fewer but bigger
(protein) particles (Figs. 3 and 4). Thus, in terms of
prevention of aggregate formation the best position for
samples is in the middle of the freezing box. This is
notwithstanding the fact that the number of particles not only
depends on position in the freezing box, but also on the
protein formulation itself. Each FT-cycle that the protein
formulation is undergoing is leading to an increase of
particle number due to repeatedly exposing the protein
to freezing/thawing stress. This phenomenon can also be
observed for the experiment series in which the protein
concentration is varied. With increased protein
concentration the number of small particles detected increases but
the amount of bigger particles decreases.
The probability of particles growing into larger ones is
smaller in sucrose solutions (
). Sucrose as a
cryoprotectant seems to have an ambivalent effect on the protein
solution in terms of particle formation. This additive is
contraproductive, i.e., induces particle formation, in
formulations con tain in g l ow protei n concentration.
However, it has the positive effect of hindering the
formation of particles for high protein concentration
(30 mg/ml and 40 mg/ml). The desired cryoprotectant
effect just occurs at higher (above 10 mg/ml) protein
concentrations wherein the sucrose protects the protein
from the ice surface. A possible reason for particle
formation in 1 mg/ml formulations could be the excess of
sucrose compared to protein concentration and hence
simply the wrong sugar/protein ratio. That is, our data
suggest there is an ideal ratio of sucrose to protein on a
U-shaped curve describing cryoprotection ability.
DLS measurements indicate a monodisperse particle
distribution in the protein formulation without sucrose regardless
of how high the protein concentration is (Table I). Also after
the addition of sucrose monodispersity remains as judged from
the low PdI. The only exception is for the 1 mg/ml protein
solution, for which the addition of sucrose increases the PdI by
a factor of 10–40. This indicates heterodispersity and
agglomeration, presumably of the sucrose itself. At the same time the
hydrodynamic diameter remains unaffected at 11.2 nm,
which corresponds to the mononomeric mAb. In other words,
the sucrose does not interact with the mAb at 1 mg/ml. By
contrast, for solutions containing 10 mg/ml or more of mAb
an increase of 2r by 1 nm ± 0.3 nm is noticed after addition of
sucrose. We rationalize this finding by a competition of water
and sucrose molecules for their presence at the protein surface
). Following the preferential exclusion mechanism (
the larger sugar molecules are excluded from the protein
surface, even though this is thermodynamically not favorable.
This results in decreased protein solubility (
) and protein
molecules being increasingly hydrated (
). In order to
compensate for the unfavorable thermodynamics the surface area
of the protein has to decrease, which is achieved by
agglomeration and a possible reason for the increase of 2r in Table I.
In other words, the presence of sucrose may destabilize the
protein’s colloidal state (
The increase of 2r upon increasing protein concentration
(Table I) is explained by the limited ability of particle diffusion
in the citrate buffer, which decreases with an increase in
concentration (»negative dynamic interaction parameter«). That
means that during freezing particles move slower (slower
diffusion) and closer together which finally leads to an increase of
hydrodynamic diameter of the aggregates due to the binding
effect of the mAb with the cosolutes present (
The OCM images show that the pure buffer, frozen at
slower rates of 1–2°C/min down to −80°C, forms multiple
ice crystals that originate from different parts of the droplet.
The see-through areas can be traced back to pure frozen
water. Due to the insolubility of salts in ice (
and other co-solutes get excluded and remain in the liquid
). The ice crystals form a freeze-front that slowly,
but progressively pushes the undisolved components as well
as the protein into the direction of crystal growth (
Channels with higher sodium citrate concentration are
formed which are visible in the images (Fig. 6). Such
concentration changes may lead to pH changes and destabilization
of the protein. (
) For faster cooling rates the ice does not
have enough time to push the solute away, so that
entrapment of the undissolvable components takes place, which
ultimately results in a more homogeneous distribution. Instead,
slower rates favor the freezing process to start from multiple
nucleation sites which leads to ice crystal growing in a
platelet–like structure orientated in various angles (Fig. 6). The
impact of sucrose is to reduce the number of nucleation
events, and thus the number of ice crystals, and at the same
time the high concentration of sucrose serves the purpose of
keeping protein molecules apart, thereby preventing
aggregation. An important finding is that sucrose addition and slow
cooling rates favor an exothermic freezing event of the FCS
over the vitrification of the FCS. That is, only with sucrose
and at slow cooling rates, also crystals form inside the veins
and interdentritic spaces, resulting in a more homogeneous
appearance in OCM images. By contrast, without sucrose or
at faster rates the FCS turns into glass, so that glassy parts and
crystalline ice co-exist. It is not entirely clear from our data
whether the second freezing event should be avoided or not –
it would be desirable to investigate the number and size of
particles for the cases of crystallization and vitrification of the
DSC results show that freezing temperatures of the sample
(buffer with and without sucrose) vary by up to 10°C, as
expected from the stochastic nature of the freezing process and
classical nucleation theory (
). The determination of Tg’ of
the freeze-concentrated sodium citrate solution had been
difficult because of the low concentration (25 mM) and limited
sensitivity of the DSC instrument. By employing faster scan
rates the signal of the DSC is intensified, which enabled us to
determine Tg’ to be −40°C at 40°C/min. The addition of
125 mM sucrose allows for detection even at slow heating
rates, and shifts Tg’ to about −32°C. This indicates about
70 wt% of sucrose in the vitrified freeze-concentrated solution.
The vitrification of the freeze-concentrated pure sodium
citrate buffer is in agreement with literature data (
). Some of
the heating curves of the pure buffer show a weak exotherm
before the main melting peak of the solution (circled area in
Fig. 9). This indicates solute crystallization, also called
coldcrystallization. Such cold-crystallization events should be
avoided to ensure product quality – which is the case after
addition of sucrose, even for slow thawing experiments.
Possibly, this is so because sucrose adsorbs on the ice surface
through hydrogen bonds between the hydroxyl groups and
the ice lattice (
), therebye preventing its crystallization.
This coating of the edges of ice might be at the origin of the
smoothing of the edges seen in Fig. 7 for slow heating
experiments. Without sucrose this can only be avoided by thawing
at rates of 20°C/min or higher. OCM images indicate that
freeze-concentrated solution melts long before ice thaws,
which enables cold-crystallization in between the ice crystals.
This is detected by the DSC and shown as a kink in the
thermograms (Fig. 9).
Tg’ of sucrose solutions is observed at around −35°C
) and shifts to higher temperatures when the heating
rate is increased (see Table II), in accordance with literature
data (68). Our data is in agreement with the data provided by
Levine and Slade, who determined Tg’ at −35°C (
emphasize that we do not see a second glass transition in our
DSC scans near −42°C, as was the case in earlier literature
). However, Fig. 8 shows that a second crystallization
process takes place upon cooling the sample beyond Tf for
sucrose-containing samples at slow cooling rates. This second
freezing event can be traced back to the solidification of
freeze-concentrated solution which has been trapped in
between the ice crystals after the first freezing event.
The freezing and thawing of pharmaceutical protein solution
has proven to be a very complex and delicate process. We
have measured particle number of protein samples after
changing parameters like composition of the buffer
formulation, sample position in the freezing box, number of FT-cycles
and protein concentration by using MFI, Archimedes and
DLS. Additionally, the buffer was analysed with and without
sucrose at different freezing and thawing rates by OCM and
DSC. Generally, in terms of particle number and hence
aggregate formation it is best to avoid multiple FT-cycles. Slow
freezing and slow thawing rates favor the formation of bigger
but fewer aggregates whereas fast freezing and fast thawing
rates lead to more, albeit smaller aggregates. The
cryoprotective-effect of sucrose is confirmed at higher protein
concentrations (above 10 mg/ml) and even then it mostly
prevents particle formation of micron and visible particles. A
reduced number of smaller particles (above 0.2 μm) could be
seen only after one FT-cycle, after 5 FT-cycles no significant
difference of particle number between formulation with and
without sucrose was detected. Besides, sucrose has been
proven to be contra-productive at 1 mg/ml protein concentrations
which is supported by data retrieved by DLS. The optimal
protein: stabilizer (sucrose) ratio has to be found in order to
achieve the desired stabilizing effect, since sucrose can lead to
conformational stabilization on one side but also
destabilization on the other side. Therefore, the optimal formulation
should improve both, solubility and kinetic stability of the
). Particle size distribution data confirm particle
concentration reduction in samples with 30 mg/ml protein
concentration. In order to determine the optimal protein
concentration regarding aggregate number reduction higher
protein concentrations have to be tested (
investigation is necessary to determine the impact of highly
concentrated mAb formulations.
OCM data support the statement that fast freezing rates
(above 30°C/min) induce microscopic scale
freeze-concentration, i.e., the solutes get trapped in between the formed ice
crystals. Also, slow freezing rates of 1–2°C/min lead to a more
uniform distribution of solutes in the frozen buffer droplet and
hence limits concentration of solutes on the microscopic scale.
The addition of sucrose shifts the melting and freezing
temperatures down by 3°C. More importantly, the addition
triggers the crystallization (rather than vitrification) of the
freezeconcentrated solution (FCS) for slow cooling rates. This
secondary freezing leads to a more homogeneous appearance of
the frozen solution. Upon heating, cold-crystallization effects
were seen to be suppressed in the presence of sucrose. The
extra exotherm around −10°C observed for buffer solutions
without sucrose until 0°C disappears when sucrose is added.
Our OCM and DSC data do not yet permit making reliable
extrapolations of cryo-concentration to the macroscopic scale
because experiments of larger volumes are requiered.
In conclusion, aggregation as a freezing-damage is
avoided best by applying slow freezing rates around 1–
2°C/min and avoiding multiple FT-cycles. Additionally,
sucrose suppresses cold-crystallization and particle
formation especially when using fast freezing rates. Higher
protein concentrations have proven to be advantageous in
terms of particle formation prevention. It has to be said
that our results apply only to our specific mAb
formulation, i.e., other mAb formulations can behave differently
and, therefore, require other handling.
ACKNOWLEDGMENTS AND DISCLOSURES
Open access funding provided by Austrian Science Fund
(FWF). We thankfully acknowledge funding by the Austrian
Science Fund FWF (bilateral project I1392) and Sandoz
GmbH, Austria/Lek Mengeš, Slovenia.
A.H. performed OCM and DSC measurements, K.P. and
D.K performed MFI, Archimedes and DLS measurements.
A.H. and T.L. wrote the paper. T.L, G.H and S.S. supervised
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License
permits unrestricted use, distribution, and reproduction in any
medium, provided you give appropriate credit to the original
author(s) and the source, provide a link to the Creative
Commons license, and indicate if changes were made.
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