The Importance of Freezing on Lyophilization Cycle Development

The Importance of Freezing
on Lyophilization Cycle
Development
Thomas W. Patapoff and David E. Overcashier
The freezing method
used during
lyophilization can
substantially affect
the structure of the
ice formed, the watervapor 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
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 residual liquid, a phase termed the freeze-concentrate.
At the point of maximal ice formation, the freezeconcentrate solidifies between the ice crystals that
make up the lattice. Under appropriate lyophilization conditions, the ice is removed by sublimation
during primary drying, leaving the remaining
freeze-concentrate in the same physical and chemical 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 sublimation) without causing cake collapse.
Less attention has been paid to the freezing
conditions and their potential effect on the primary and secondary drying processes and on the
characteristics of the final product. Kochs et al.
reported the effects of freezing conditions on primary drying in a specially designed aluminum
and plastic sample cell (10). They observed variations 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 annealing step in the freezing procedure increased
the drying rate (12).
Our article describes the effects of several
freezing methods on water-vapor flow during primary 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. Typically, the shelf temperature is then decreased (from
5 °C) over three hours and held at a low temperature (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 typically occurring at 15 °C. Freezing point depression accounts for a lowering of the freezing temperature by only 1.8 kelvin per molal. Most
pharmaceutical manufacturing relies on supercooling 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.
MASS TRANSFER RESISTANCE
An important parameter governing the relationship between independent variables (the shelf
temperature and chamber pressure) and dependent 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 proportionality 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 crosssectional area of the product in the vial, MTR is
the normalized dried product resistance, Po is the
equilibrium vapor pressure of ice at the temperature 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 (electrical 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 sequentially stoppering the vials during primary drying,
Freezing
Method
Shelf
Temp (°C)
Sublimation
Rate (g/hr)
Product
Temp (°C)
Standard
Vertical
Vertical
Vertical
10
10
20
30
0.36
0.46
0.48
0.55
24.5
27.0
26.0
23.5
Dried Layer
[Resistance (cm2 mTorr hr g1)]
(a)
10,000
Table 1. Effect of freezing
method and primary drying shelf
temperature on sublimation rate
and primary drying product
temperature
Fast freezing
Standard freezing
Vertical freezing
Annealed
8,000
6,000
4,000
2,000
0
(b)
Derivative
[Resistance (cm2 mTorr hr g1)]
However, the entire solution cannot freeze immediately because the supercooled water can absorb
only 15 cal/g of the 79 cal/g of heat given off by
the ice formation. Therefore, the ice propagates
from the nucleation site, and about 19% crystallizes in multibranching, tortuous paths while the
vial contents warm to approximately 1 °C.
After the initial ice network has formed, additional heat is removed from the solution by the
shelf, and the remaining water freezes when the
previously formed ice crystals grow.
Primary drying. At the completion of freezing,
primary drying is initiated with the lowering of
the chamber pressure and an increase in the shelf
temperature. The ice sublimation front moves
downward through the frozen plug, a process that
makes the water vapor transit through the channels left by the sublimed ice above. Those narrow,
often tortuous channels resist vapor flow, so sublimation is slowed. Consideration of the vapor
channels illustrates how the method of freezing,
by affecting ice crystal structure, can affect
primary drying. Decreasing the resistance to mass
transfer of water vapor increases the sublimation
rate, which leads to shorter primary drying times
while lowering the product temperature.
50,000
Fast freezing
Standard freezing
Vertical freezing
Annealed
40,000
30,000
20,000
10,000
0
0
0.2
0.4
0.6
0.8
1.0
Dried Layer Thickness (cm)
Figure 1. Mass transfer
resistance as a function of dried
layer thickness for material
frozen by various methods: (a)
cumulative MTR, (b) derivative
of MTR
then weighing the amount of the remaining solution. We previously published a complete description 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 (increasing the thickness of the dried layer). That indicates 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
image under a
fluorescence
microscope
lyophilize warm, add black paraffin
under vacuum
cut plug
cool,
release
plug
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 developed (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 paraffin 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 channels 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, tortuous 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 thickness 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 direction of the channels near the surface of the cake.
The restricted vapor flow results in slower ice sublimation.
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 channel orientation and the lack of thick skin on top
lowers the MTR from that of samples from standard 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
bottom, because the local MTR values are equivalent for dried layer thickness greater than 0.5 cm.
Vertical freezing produces a higher sublimation rate and lower product temperature during
primary drying (Table 1). The lower MTR allows
faster sublimation, which results in lower product
temperatures through increased heat removal
from the latent heat of sublimation. The vertical
freezing results are consistent with those found
by Searles et al. that the presence of lamellar
(instead of spheroidal) ice crystals increases drying rates (11).
Figure 3. Freeze-dried cake
appearance corresponding to
standard and alternative
freezing methods:
(a) supercooling using standard
freezing; (b) fast freezing;
(c) vertical freezing; and
(d) standard freezing followed
by annealing (photo courtesy of
Genentech, Inc.)
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 spontaneous 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
solutions frozen by the standard or fast processes
and is comparable to solutions that were nucleated on the bottom with vertical ice propagation.
The lower resistance observed for annealed
material is consistent with a faster sublimation
reported by Searles et al. (12). Figure 1b also
shows that the annealed material has a local MTR
that is low and constant throughout the cake. The
effects of any skin appear to be minimal.
DECREASING MTR BY OTHER METHODS
Depending on solution composition, it may be
possible to adjust the temperature (or the formulation) such that primary drying takes place at
product temperatures near the glass transition
temperature (Tg) of the freeze-concentrate. That
allows the freeze-concentrate to flow enough to
create holes in the channel walls so water vapor
can be transmitted in a less tortuous path, more
directly up and out of the cake.
Figure 4a shows an SEM result in which holes
have formed in the walls of the pores. These
porous walls were not evident in formulations
containing protein (Figure 4b) but were observed
only in formulations using a protein-free placebo,
which has a lower Tg. The holes were less
evident in the protein-free material when it was
dried at lower temperatures. That implies that not
all formulations are amenable to pore formation.
Figure 5 shows that the MTR for the placebo
was significantly lower than that of the solution
that contains protein (9). Obviously, the selection
of processing conditions needs to consider final
product quality. The possibility that such a smallscale collapse might alter the molecular structure
or stability of the product would need to be evaluated by an appropriate analytical program.
Table 2. Effect of freezing
method on the dried state
stability of a therapeutic proteina
Freezing
Method
Standard
Annealed
Aggregation
Rateb
2.3c
1.6
a2.5 mg/mL protein, 123 mg/mL
total excipient
bat 50 °C
cpercent per month
OPTIMIZING THE CYCLE
The manner of freezing can have a significant
impact on primary drying characteristics of a
formulation. For a given primary drying shelf
temperature and chamber pressure, a decrease in
MTR will increase the rate of sublimation. In
addition, an increase in the rate of sublimation
will lead to a lower product temperature because
of the enthalpy of ice sublimation (Table 1).
Increasing the primary drying shelf temperature of
vertically frozen vials increases the product tem-
BioPharm
MARCH 2002
19
Dried Layer
[Resistance (cm2 mTorr hr g1)]
6,000
Active
Placebo
5,000
4,000
3,000
2,000
1,000
0
0
0.2 0.4 0.6
0.8
1
Dried layer thickness (cm)
Figure 5. Cumulative mass transfer resistance as
a function of dried layer thickness for active and
placebo material
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,
[email protected], www.gene.com.
20
BioPharm
MARCH 2002
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 temperatures, the rate of sublimation was 40% faster for
vertically oriented ice than for standard-frozen solutions (Table 1). That leads to a decrease in the
time required for primary drying — which constitutes 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 consequences 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 decrease 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 ability to stabilize proteins in the dried state (27–30).
Some proteins undergo cold denaturation during
slow freezing or annealing, which can have deleterious 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
overcome by standard processes. Ice might be
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 variations 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 stability for some proteins. Understanding and controlling 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.
REFERENCES
(1) S.L. Nail, “The Effect of Chamber Pressure on Heat
Transfer in the Freeze Drying of Parenteral
Solutions,” PDA J. Pharm. Sci. Technol. 34,
358–368 (1980).
(2) M.J. Pikal et al., “Physical Chemistry of FreezeDrying: Measurement of Sublimation Rates for
Frozen Aqueous Solutions by a Microbalance
Technique,” J. Pharm. Sci. 72, 635–650 (1983).
(3) M.J. Pikal, M.L. Roy, and S. Shah, “Mass and
Heat Transfer in Vial Freeze-Drying of
Pharmaceuticals: Role of the Vial,” J. Pharm.
Sci. 73, 1224–1237 (1984).
(4) M.J. Pikal, “Use of Laboratory Data in Freeze
Drying Process Design: Heat and Mass
Transfer Coefficients and the Computer
Simulation of Freeze Drying,” PDA J.
Parenteral Sci. Technol. 39, 115–139 (1985).
(5) R.G. Livesey and T.W.G. Rowe, “A
Discussion of the Effect of Chamber Pressure
on Heat and Mass Transfer in Freeze-Drying,”
J. Parenteral Sci. Technol. 41, 169–171
(1987).
(6) M.J. Pikal and S. Shah, “The Collapse
Temperature in Freeze Drying: Dependence on
Measurement Methodology and Rate of Water
Removal from the Glassy Phase,”
Int. J. Pharm. 62, 165–186 (1990).
(7) M.J. Pikal, “Freeze-Drying of Proteins, Part 1:
Process Design,” BioPharm 3(8), 18–27 (1990).
(8) B.S. Chang and N.L. Fischer, “Development
of an Efficient Single-Step Freeze-Drying
Cycle for Protein Formulations,” Pharm. Res.
12, 831–837 (1995).
(9) D.E. Overcashier, T.W. Patapoff, and C.C. Hsu,
“Lyophilization of Protein Formulations in
Vials: Investigation of the Relationship Between
Resistance to Vapor Flow During Primary
Drying and Small-Scale Product Collapse,” J.
Pharm. Sci., 88, 688–695 (1999).
(10) M. Kochs et al., “The Influence of the
Freezing Process on Vapour Transport During
Sublimation in Vacuum-Freeze-Drying of
Macroscopic Samples,” Int. J. Heat Mass
Transfer, 36, 1727–1738 (1993).
(11) J.A. Searles, J.F. Carpenter, and T.W.
Randolph, “The Ice Nucleation Temperature
(12)
(13)
(14)
(15)
(16)
(17)
Determines the Primary Drying Rate of
Lyophilization for Samples Frozen on a
Temperature-Controlled Shelf,”
J. Pharm. Sci., 90(7), 860–871 (2001).
J.A. Searles, J.F. Carpenter, and T.W.
Randolph, “Annealing to Optimize the Primary
Drying Rate, Reduce Freezing-Induced Drying
Rate Heterogeneity, and Determine Tg in
Pharmaceutical Lyophilization,” J. Pharm. Sci.
90, 872–887 (2001).
G. Jancso, J. Pupezin, W.A. Van Hook, “The
Vapor Pressure of Ice Between +10-2 and
10+2,” J. Phys. Chem. 74, 2984–2989
(1970).
L. Gatlin and P.P. DeLuca, “A Study of the
Phase Transitions in Frozen Antibiotic
Solutions by Differential Scanning
Calorimetry,” PDA J. Pharm. Sci. Technol.,
34, 398–408 (1980).
E. Tarelli et al., “Additives to Biological
Substances III: The Moisture Content and
Moisture Uptake of Commonly Used Carrier
Agents Undergoing Processing Conditions
Similar to Those Used in the Preparation of
International Biological Standards,”
J. Biol. Stand., 15, 331–340 (1987).
P.J. Dawson and D.J. Hockley, “Scanning
Electron Microscopy of Freeze-Dried
Preparations: Relationship of Morphology to
Freeze-Drying Parameters,” International
Symposium on Biological Product FreezeDrying Parameters, Bethesda, MD.
Developments in Biological Standards, Vol. 74
(S. Karger AG, Basel, Switzerland, 1990),
pp.185–192.
N. Murase, P. Echlin, and F. Franks, “The
Structural States of Freeze-Concentrated and
Info #16
(18)
(19)
(20)
(21)
(22)
(23)
(24)
Freeze-Dried Phosphates Studied by Scanning
Electron Microscopy and Differential
Scanning Calorimetry,” Cryobiology, 28,
364–375 (1991).
B.S. Chang and C.S. Randall, “Use of
Subambient Thermal Analysis to Optimize
Protein Lyophilization,” Cryobiology, 29,
632–656 (1992).
R. Haikala et al., “Polymorphic Changes of
Mannitol During Freeze-Drying: Effect of
Surface-Active Agents,” PDA J. Pharm. Sci.
Technol., 51, 96–101 (1997).
N. Milton et al., “Evaluation of Manometric
Temperature Measurement as a Method of
Monitoring Product Temperature During
Lyophilization,” PDA J. Pharm. Sci. Technol.,
51, 7–16 (1997).
L. van den Berg and D. Rose, “Effect of
Freezing on the pH and Composition of
Sodium and Potassium Phosphate Solutions:
The Reciprocal System KH2PO4-Na2HPO4H2O,” Arch. Biochem. Biophys., 81, 319–329
(1959).
N. Murase and F. Franks, “Salt Precipitation
During the Freeze-Concentration of Phosphate
Buffer Solutions,” Biophys. Chem., 34,
293–300 (1989).
G. Gomez, M.J. Pikal, and N. RodriguezHornedo, “Effect of Initial Buffer Composition
on pH Changes During Far-from-Equilibrium
Freezing of Sodium Phosphate Buffer
Solutions,” Pharm. Res., 18, 90–97 (2001).
T.J. Anchordoquy and J.F. Carpenter,
“Polymers Protect Lactate Dehydrogenase
During Freeze-Drying by Inhibiting
Continued on page 72
BioPharm
MARCH 2002
21
Validation
Importance of Freezing continued from page 21
(25)
(26)
(27)
(28)
72
Dissociation in the Frozen State,” Arch.
Biochem. Biophys., 332, 231–238 (1996).
X.M. Lam et al., “Replacing Succinate with
Glycolate Buffer Improves the Stability of
Lyophilized Interferon-,” Int. J. Pharm., 142,
85–95 (1996).
K.A. Pikal-Cleland et al., “Protein
Denaturation During Freezing and Thawing in
Phosphate Buffer Systems: Monomeric and
Tetrameic -Galactosidase,” Arch. Biochem.
Biophys., 384, 398–406 (2000).
J.F. Carpenter, S.J. Prestrelski, and T.
Arakawa, “Separation of Freezing- and
Drying-Induced Denaturation of Lyophilized
Proteins Using Stress-Specific Stabilization,
I: Enzyme Activity and Calorimetric Studies,”
Arch. Biochem. Biophys., 303, 456–464
(1993).
K.-i. Izutsu, S. Yoshioka, and T. Terao,
“Decreased Protein-Stabilizing Effects of
Cryoprotectants Due to Crystallization,”
Pharm. Res., 10, 1232–1237 (1993).
BioPharm
MARCH 2002
(29) H.R. Costantino et al., “Effect of Excipients on
the Stability and Structure of Lyophilized
Recombinant Human Growth Hormone,”
J. Pharm. Sci., 87, 1412–1420 (1998).
(30) L. Kreilgaard et al., “Effects of Additives on
the Stability of Humicola Lanuginosa Lipase
During Freeze-Drying and Storage in the Dried
Solid,” J. Pharm. Sci., 88, 281–290 (1999).
(31) C.C. Hsu et al., “Surface Denaturation at
Solid–Void Interface: A Possible Pathway by
Which Opalescent Particulates Form During
the Storage of Lyophilized Tissue-Type
Plasminogen Activator at High Temperatures,”
Pharm. Res., 12, 69–77 (1994).
(32) B.S. Chang, B.S. Kendrick, and J.F. Carpenter,
“Surface-Induced Denaturation of Proteins
During Freezing and Its Inhibition by
Surfactants,” J. Pharm. Sci., 85, 1325–1330
(1996).
(33) S. Jiang and S.L. Nail, “Effect of Process
Conditions on Recovery of Protein Activity
After Freezing and Freeze-Drying,” Eur. J.
Pharma. Biopharma., 45, 249–257 (1998). BP