The Importance of Freezing
on Lyophilization Cycle
The freezing method
used during
lyophilization can
substantially affect
the structure of the
ice formed, the water-
vapor flow during
primary drying, and
the quality of the final
dried product.
Controlling how a
solution freezes can
shorten lyophilization
cycles and produce
more stable
formulations.
16 BioPharm MARCH 2002
Development
Thomas W. Patapoff and David E. Overcashier
L
yophilization (freeze-drying) is often
used to prepare dry pharmaceutical
formulations to achieve commercially
viable shelf lives. The process
comprises three steps: freezing, primary
drying, and secondary drying. As water
freezes in the first step, the dissolved
components in the formulation remain in the resid-
ual liquid, a phase termed the freeze-concentrate.
At the point of maximal ice formation, the freeze-
concentrate solidifies between the ice crystals that
make up the lattice. Under appropriate lyophiliza-
tion conditions, the ice is removed by sublimation
during primary drying, leaving the remaining
freeze-concentrate in the same physical and chemi-
cal structure as when the ice was present. Residual
water in the freeze-concentrate is removed in the
secondary drying step.
Lyophilization cycle development typically
focuses on optimizing the primary drying step.
That is the most time consuming of the three
steps, and the primary drying parameters are
easily adjustable. They can affect both the time
involved and the quality of the resulting cake.
Extensive investigation of primary drying has
demonstrated that two important parameters are
chamber pressure and shelf temperature (1–9).
They are usually adjusted to maximize the rate of
heat transfer to each vial (speeding ice sublima-
tion) without causing cake collapse.
Less attention has been paid to the freezing
conditions and their potential effect on the pri-
mary and secondary drying processes and on the
characteristics of the final product. Kochs et al.
reported the effects of freezing conditions on pri-
mary drying in a specially designed aluminum
and plastic sample cell (10). They observed varia-
tions in vapor diffusion coefficients (a measure of
the ease of water-vapor flow) as a function of
position and cooling rate. The variations
appeared to be largely due to variations in sample
morphology. Searles et al. reported some effects
of freezing on the rate of primary drying in vials
(11). They found that the primary drying rate was
dependent on the ice nucleation temperature.
Faster drying was also attributed to an increase in
lamellar ice crystal content, formed when ice
nucleants from Pseudomonas syringae were
added in the vial contents. Searles and colleagues
also showed that adding a properly specified an-
nealing step in the freezing procedure increased
the drying rate (12).
Our article describes the effects of several
freezing methods on water-vapor flow during pri-
mary drying and on the final dried cake structure.
NORMAL FREEZING AND SUBLIMATION
When freeze-drying pharmaceutical products,
manufacturers place vials containing aqueous
formulations on shelves within a lyophilizer. Typi-
cally, the shelf temperature is then decreased (from
5 °C) over three hours and held at a low tempera-
ture (typically 40 °C to 50 °C) to ensure that
all the vials freeze completely. As the temperature
decreases, the liquid cools below the freezing point
of the solution: Supercooled temperatures range
from 10 °C to 20 °C with ice nucleation typi-
cally occurring at 15 °C. Freezing point depres-
sion accounts for a lowering of the freezing tem-
perature by only 1.8 kelvin per molal. Most
pharmaceutical manufacturing relies on supercool-
ing because of the lack of nucleation centers in the
formulations (such as particulates in solution or
imperfections on the inside surface of the vials).
In a supercooled solution at 15 °C, ice can
nucleate and propagate through the solution.
However, the entire solution cannot freeze imme- Table 1. Effect of freezing
diately because the supercooled water can absorb method and primary drying shelf
Freezing Shelf Sublimation Product
only 15 cal/g of the 79 cal/g of heat given off by Method Temp (°C) Rate (g/hr) Temp (°C) temperature on sublimation rate
the ice formation. Therefore, the ice propagates and primary drying product
Standard 10 0.36 24.5
from the nucleation site, and about 19% crystal- Vertical 10 0.46 27.0 temperature
lizes in multibranching, tortuous paths while the Vertical 20 0.48 26.0
vial contents warm to approximately 1 °C. Vertical 30 0.55 23.5
After the initial ice network has formed, addi-
tional heat is removed from the solution by the
shelf, and the remaining water freezes when the
previously formed ice crystals grow. (a) Fast freezing
Primary drying. At the completion of freezing,

[Resistance (cm2 mTorr hr g1)]

Standard freezing
10,000 Vertical freezing
primary drying is initiated with the lowering of
Annealed
the chamber pressure and an increase in the shelf 8,000

Dried Layer
temperature. The ice sublimation front moves
6,000
downward through the frozen plug, a process that
makes the water vapor transit through the chan- 4,000
nels left by the sublimed ice above. Those narrow,
often tortuous channels resist vapor flow, so subli- 2,000

mation is slowed. Consideration of the vapor 0

channels illustrates how the method of freezing,
by affecting ice crystal structure, can affect (b)
[Resistance (cm2 mTorr hr g1)]

primary drying. Decreasing the resistance to mass 50,000

transfer of water vapor increases the sublimation Fast freezing
rate, which leads to shorter primary drying times 40,000
Standard freezing
Derivative

while lowering the product temperature. Vertical freezing

30,000 Annealed

MASS TRANSFER RESISTANCE 20,000

An important parameter governing the relation-
ship between independent variables (the shelf
temperature and chamber pressure) and depen-
dent variables (the ice sublimation rate and the
product temperature) has been defined by Pikal
as the resistance to water-vapor flow or the mass
transfer resistance (MTR) (2).
Determining MTR. MTR describes the propor-
tionality between the specific sublimation rate
m and the pressure driving force PoPv:
Ap
Po –Pv
MTR =
m /A p
where m is the sublimation rate, Ap is the cross-
sectional area of the product in the vial, MTR is
the normalized dried product resistance, Po is the
equilibrium vapor pressure of ice at the tempera-
ture of the subliming ice, and Pv is the pressure in
the chamber. The relationship is analogous to that
of electrical resistance: For a given pressure
driving force (voltage), a decrease in MTR (elec-
trical resistance) increases the rate of sublimation
(electrical current). To determine MTR, measure
the product temperature (from which we obtain Po
using the equation published by Jancso et al., 13)
and the sublimation rate m . These measurements
can be obtained using a thermocouple and sequen-
tially stoppering the vials during primary drying,
Figure 1. Mass transfer
10,000
resistance as a function of dried
0 layer thickness for material
0 0.2 0.4 0.6 0.8 1.0 frozen by various methods: (a)
Dried Layer Thickness (cm) cumulative MTR, (b) derivative
of MTR
then weighing the amount of the remaining solu-
tion. We previously published a complete descrip-
tion of this method for determining MTR (9).
Effect of the freezing method on MTR. Figure 1
shows MTRs during primary drying (10 °C shelf
temperature, 100 mTorr chamber pressure) for
vials frozen using the standard freezing process
that we described earlier and for those frozen by
alternative methods described below (plots used
25 mg/mL rhuMAb HER2, 20 mg/mL trehalose,
0.1 mg/mL polysorbate 20, and 5 mM histidine,
with a 6.0 pH, filled at 5.0 mL in 10cc tubing
vials). Following standard freezing, the resistance
initially rose quickly, then more slowly as the
sublimation front moved down the vial (increas-
ing the thickness of the dried layer). That indi-
cates that the upper layer of the dried cake
contributed more to the resistance than did the
lower portion, suggesting that the structure of the
dried layer varied in the vertical direction.
Curves in Figure 1a represent the effect of the
dried layer thickness on the cumulative MTR. The
BioPharm MARCH 2002 17

filled vial add fluorescent dye lyophilize warm, add black paraffin
under vacuum
cool,
image under a release
fluorescence plug
cut plug
microscope
Figure 2. Paraffin dye method
local MTR for various cake depths can be
determined from the derivative of those curves
(Figure 1b). The derivative curve (for standard
freezing) also shows that the upper layer of the
dried cake contributes more to the resistance than
does the lower portion. That result is consistent
with the results published by Kochs et al. (10).
The relationship between resistance and dried
cake structure can be investigated by studying
cake structure after lyophilization.
CAKE STRUCTURE
The structure of a lyophilized cake has been
reported several times (14–20). In most cases,
cakes came from a supercooled solution with little
or no attempt at controlling ice nucleation or
propagation direction. Samples were generally cut
or pulled apart and viewed by scanning electron
microscopy (SEM). Because the potentially fragile
cake structure was unsupported, the area exposed
may have been altered by the cutting. If the cake is
pulled apart, the tear will likely follow a structural
weakness in the cake, so the exposed area may not
be representative of the rest of the cake.
In the paraffin investment technique we devel-
oped (Figure 2), paraffin acts as a support for the
physical fine structure of the cake and also
prevents water absorption during sectioning,
observation, and storage (9). The method uses a
fluorescent molecule (such as rhodamine B) that
is added to the solution before lyophilization and
distributes equally throughout the cake (with no
visible phase separation or spatial partitioning).
After lyophilization, the cake in the vial is placed
under vacuum and warmed to 55 °C. Then paraf-
fin is poured into the vial. While warm and under
18 BioPharm MARCH 2002
vacuum, the paraffin enters all the pores of the
cake with no observable disturbance to the cake
structure. The vial is then removed from the
vacuum oven, cooled, and sectioned. The
sectioned cakes are then observed under a
fluorescence microscope. Interference from the
fluorescence deeper in the cake is minimized by
using a dark dye in the paraffin.
Correlating MTR to cake structure. The tortuous
path that supercooled ice takes when propagating
(Figure 3a) shows little directionality to the chan-
nels after sublimation. Those channels, roughly
25–100 m in diameter, serve as conduits for
water-vapor transport from the sublimation front
to the vial headspace. Resistance to vapor flow
out of the cake is a result of both the small, tortu-
ous channels and thick skin-like material on the
top of the cake (reaching a thickness greater than
1–2 mm). In the standard freezing scenario of
Figures 1a and 1b, high MTR can be seen as the
sublimation front moves down through the first
0.2 cm of the cake, corresponding to the thick-
ness of the skin. Once the sublimation front
passed the skin, the cumulative MTR increased
more slowly (suggesting lower local MTR).
CONTROLLING ICE NUCLEATION
One way to produce directional freezing is to
freeze the vial contents rapidly by immersing the
vial into a dry-ice/ethanol bath until it is completely
frozen. The resulting pores left from the rapid
freezing and ice propagation are very small
(Figure 3b). They start at the walls of the vial,
move toward the middle, then move up. Although
directional, the pores are small and nonvertical.
The effect of such fast freezing can be seen in the
MTR: The cumulative MTR increases rapidly and
to a higher level (Figure 1a) and shows very high
local MTR at small cake depths (Figure 1b). For
fast freezing, that high MTR is attributable to the
small size of the pores and to the horizontal direc-
tion of the channels near the surface of the cake.
The restricted vapor flow results in slower ice sub-
limation.
Vertical freezing. To obtain straight, vertical
channels, solutions were cooled on wet ice,
nucleated at the bottom with dry-ice, and placed
on a 50 °C shelf. The ice crystals propagate
vertically, and no thick skin forms on the cake
surface (Figure 3c). As expected, a vertical chan-
nel orientation and the lack of thick skin on top
lowers the MTR from that of samples from stan-
dard and fast-freezing protocols (Figure 1a).
Figure 1b shows that the local MTR is also low
and constant for all cake depths. Interestingly,
Figure 1b also suggests that changing the freezing
method did not alter the cake structure at the vial Figure 3. Freeze-dried cake
bottom, because the local MTR values are equiva- appearance corresponding to
lent for dried layer thickness greater than 0.5 cm. standard and alternative
Vertical freezing produces a higher sublima- freezing methods:
tion rate and lower product temperature during (a) supercooling using standard
primary drying (Table 1). The lower MTR allows freezing; (b) fast freezing;
faster sublimation, which results in lower product (c) vertical freezing; and
temperatures through increased heat removal (d) standard freezing followed
from the latent heat of sublimation. The vertical by annealing (photo courtesy of
freezing results are consistent with those found Genentech, Inc.)
by Searles et al. that the presence of lamellar
(instead of spheroidal) ice crystals increases dry-
ing rates (11).

SUPERCOOLING AND ANNEALING

Ice structure and MTR during primary drying can
be altered by freezing the vial contents using the
standard method (supercooling followed by spon-
taneous nucleation), then warming the frozen
solution in the vial to a temperature just below
the melting point (for example 2 °C) for
10 hours. This allows the ice crystals to rearrange
and grow to a more stable state. The vials are
then cooled again to 50 °C. Figure 3d shows
results from that annealing process. The cakes
produced after annealing tend to have larger
pores than those from the standard freezing
method, which would be expected to facilitate
water-vapor transmission and lower the MTR
during primary drying. Figure 1a shows that the
annealed material has a much lower MTR than
only in formulations using a protein-free placebo, Table 2. Effect of freezing
solutions frozen by the standard or fast processes
which has a lower Tg. The holes were less method on the dried state
and is comparable to solutions that were nucle-
evident in the protein-free material when it was stability of a therapeutic proteina
ated on the bottom with vertical ice propagation.
dried at lower temperatures. That implies that not
The lower resistance observed for annealed
all formulations are amenable to pore formation.
material is consistent with a faster sublimation Freezing Aggregation
Figure 5 shows that the MTR for the placebo Method Rateb
reported by Searles et al. (12). Figure 1b also
was significantly lower than that of the solution
shows that the annealed material has a local MTR Standard 2.3c
that is low and constant throughout the cake. The that contains protein (9). Obviously, the selection Annealed 1.6
effects of any skin appear to be minimal. of processing conditions needs to consider final a2.5 mg/mL protein, 123 mg/mL
product quality. The possibility that such a small- total excipient
scale collapse might alter the molecular structure bat 50 °C
DECREASING MTR BY OTHER METHODS cpercent per month
Depending on solution composition, it may be or stability of the product would need to be eval-
possible to adjust the temperature (or the formu- uated by an appropriate analytical program.
lation) such that primary drying takes place at
product temperatures near the glass transition OPTIMIZING THE CYCLE
The manner of freezing can have a significant
temperature (Tg) of the freeze-concentrate. That
impact on primary drying characteristics of a
allows the freeze-concentrate to flow enough to
formulation. For a given primary drying shelf
create holes in the channel walls so water vapor
temperature and chamber pressure, a decrease in
can be transmitted in a less tortuous path, more
MTR will increase the rate of sublimation. In
directly up and out of the cake.
addition, an increase in the rate of sublimation
Figure 4a shows an SEM result in which holes
will lead to a lower product temperature because
have formed in the walls of the pores. These
of the enthalpy of ice sublimation (Table 1).
porous walls were not evident in formulations
Increasing the primary drying shelf temperature of
containing protein (Figure 4b) but were observed
vertically frozen vials increases the product tem-
BioPharm MARCH 2002 19

Figure 4. SEM micrographs of
freeze-dried material and the
effect of formulation on
microstructure: (a) placebo
(45 mg/mL trehalose,
0.1 mg/mL polysorbate 20, and
5 mM histidine at 6.0 pH) and
(b) active protein (25 mg/mL
rhuMAb HER2, 20 mg/mL
trehalose, 0.1 mg/mL
polysorbate 20, and 5 mM
histidine at 6.0 pH) (photo
courtesy of Genentech, Inc.)
Corresponding author Thomas W.
Patapoff is a senior scientist and
director, and David E. Overcashier
is a senior process engineer in
Pharmaceutical R&D at
Genentech, Inc., One DNA Way,
South San Francisco, CA 94080,
650.225.8006, fax 650.225.7234,
tomp@gene.com, www.gene.com.
20 BioPharm MARCH 2002
[Resistance (cm2 mTorr hr g1)]
6,000
5,000 Active
Placebo
Dried Layer
4,000
3,000
2,000
1,000
0 overcome by standard processes. Ice might be
0 0.2 0.4 0.6 0.8
Dried layer thickness (cm)
Figure 5. Cumulative mass transfer resistance as
a function of dried layer thickness for active and
placebo material
perature for primary drying, which then matches
that of a standard-frozen material dried at a colder
shelf temperature. That higher shelf temperature
adds driving force to the heat transfer, speeding
primary drying. For equivalent product tempera-
tures, the rate of sublimation was 40% faster for
vertically oriented ice than for standard-frozen so-
lutions (Table 1). That leads to a decrease in the
time required for primary drying — which consti-
tutes half of the time spent in the lyophilization
process. Such a decrease should also be expected
from annealed vials, so the duration of the
lyophilization cycle should be reduced as well.
OTHER CONSEQUENCES OF FREEZING
The freezing process can have unexpected conse-
quences on product quality. For example, some
forms of sodium phosphate can crystallize upon
slow freezing or annealing, resulting in a pH
decrease in the freeze-concentrate (21–23). A de-
crease in pH has been shown to affect the stability
of a drug when frozen and after lyophilization
(24–26). Other excipients that crystallize during
freezing (for example, mannitol) can lose their abil-
ity to stabilize proteins in the dried state (27–30).
Some proteins undergo cold denaturation during
slow freezing or annealing, which can have delete-
rious effects on product quality upon reconstitution.
Freezing methods can alter the void–solid
interfacial area of a lyophilized cake and,
inversely, the thickness of its channel walls. An
increased surface area correlates with decreased
dried product stability for some proteins (31).
Freezing by immersing a vial into liquid nitrogen
can result in increased protein aggregation (32) or
decreased enzyme activity (33) when compared
with freezer-cooled samples. Preliminary studies
indicate that the freezing method has a significant
effect on dried protein stability (Table 2). In addi-
tion, reconstitution time could be affected
because increasing the surface area may allow
faster reconstitution.
PRACTICAL ASPECTS
Freezing methods are seldom investigated
because freezing is difficult to control. Normally,
pharmaceutical products supercool to roughly
15 °C before nucleating, which is difficult to
1
induced to nucleate at higher temperatures (closer
to 0 °C) by conditioning the bottom surface of the
glass vial. Nucleation would then occur on the
bottom surface, and the ice would propagate in a
vertical direction resulting in lower MTRs. Vials
with that characteristic are not yet available.
Annealing can be more practical but also has
limitations. Whether annealing influences the
time for lyophilization or the temperature varia-
tions of the product during freezing needs to be
demonstrated.
Shortening the cycle. The freezing method used
during lyophilization can have a significant effect
on the structure of the ice formed, affecting both
the water-vapor flow during primary drying and
the final product. Freezing protein solutions in
vials with vertically propagated crystals may
prevent a thick skin from forming on the top of
the lyophilized cake. The structure of vertically
frozen material facilitates fast water-vapor flow
during primary drying, resulting in lower product
temperatures. By increasing the primary drying
shelf temperature, the rate of sublimation was
40% faster in our studies for the vertically frozen
material than for the standard-frozen material
with an equivalent product temperature. We also
observed increased sublimation in material
annealed during the freezing segment. Decreased
surface area correlates with increased product sta-
bility for some proteins. Understanding and con-
trolling how a solution freezes can lead to shorter
lyophilization cycles and, in some cases, more
stable products.
ACKNOWLEDGMENTS
The authors are indebted to Jamie Moore, Mary
Cromwell, Anne Nguyen, Al Stern, and Chung Hsu for
technical assistance and consultation.
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