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An Improved Method for Visualizing the Morphology of

Lyophilized Product Cakes
Philippe Lam and Thomas W. Patapoff

PDA J Pharm Sci and Tech 2011, 65 425-430

Access the most recent version at doi:10.5731/pdajpst.2011.00749
 Downloaded from on June 21, 2021

Technology/Application

An Improved Method for Visualizing the Morphology of

Lyophilized Product Cakes
PHILIPPE LAM1 and THOMAS W. PATAPOFF2,*
1
Late Stage Pharmaceutical Development and 2Early Stage Pharmaceutical Development, Genentech, Inc. ©PDA,
Inc. 2011

ABSTRACT: Due to low optical contrast, the morphology of lyophilized product cakes is difficult to observe and
photograph. Furthermore, internal structures are normally not visible unless the cake is fractured. Because most
lyophilized substances are hygroscopic and quite fragile, the product cake, once removed from the vial, will rapidly
degrade. We propose herein a technique that allows a lyophilized product cake to be preserved, manipulated, and
easily observed outside the vial. This technique yields high-quality, cross sectional images that reveal intricate fine
structures without the use of expensive specialized equipment.

KEYWORDS: Lyophilization, Visualization, Cake, Structure, Encapsulation, Freeze-drying

Introduction Previously, Patapoff and Overcashier (1) introduced a

During lyophilization cycle development or investiga-
tions of production lyophilization process deviations,
a visual inspection of the lyophilizate cake is generally
performed as part of the assessment. Particular cake
features can provide some clues to events that trans-
pired during the freeze drying operation. For example,
a cake that exhibits regions of partial collapse can be
indicative of excessive shelf temperature during the
primary drying step. Frequently, these regions of par-
tial collapse cannot be seen by external inspection
while the the cake remains in the vial.
It is desirable to maintain a photographic record of the
various lyophilizate cakes for documentation purpose.
However, properly imaging a low-contrast sample
through the highly curved surface of a glass vial presents
significant challenges. Extracting the cake from the vial
can improve the quality of the final images and allow for
the observation of internal structures by sectioning with
a sharp blade. However, due to their fragile nature, the
manipulation of naked cakes is difficult and, for hygro-
scopic products, the cakes may also “melt” rapidly once
exposed to ambient conditions.
* Corresponding author: Thomas W. Patapoff, Genen-
tech, Inc., 1 DNA Way, South San Francisco, CA
94080, (650) 225-8006, tomp@gene.com
doi: 10.5731/pdajpst.2011.00749
technique that involved embedding a lyophilizate cake
in a low-melting-temperature paraffin. Once solidified,
the cake-containing paraffin plug could be removed by
breaking the vial. The authors have shown that even
microscopic, intricate structures can be easily photo-
graphed and preserved for a substantial length of time
in this fashion. There are, however, several require-
ments, such as the need to incorporate a fluorescent
dye to the liquid prior to freeze-drying and the expo-
sure of the final cake to a temperature of ⬃60 °C,
which limit the utility of the technique to lyophiliza-
tion runs carried in the laboratory and to cakes that can
withstand higher temperatures without significant
structural change.
In this paper we present an improved technique that is
not subject to the limitations mentioned above and that
produces comparable image quality. The new proce-
dure still involves encapsulation of the lyophilizate
cake and a fluorescent dye for imaging, but in this
instance a silicone elastomer is used as the matrix and
the dye is added just before visual observation. Prod-
uct vials from both laboratory and large-scale produc-
tion facilities have been successfully processed and
imaged in this manner.
For the purpose of illustration, we have encapsulated
and imaged lyophilized samples that had been frozen
in a controlled manner and show that the resulting
cake morphologies are as expected (1, 2).

Vol. 65, No. 4, July–August 2011 425

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Material and Methods
Preparation of Lyophilized Samples
Two solutions were used to demonstrate the usefulness
of the embedding and imaging technique. One solution
(with no protein) contained 4% trehalose, 50 mM
sodium phosphate at pH 6.2, and 0.02% polysorbate
20. A second solution contained 25 mg/mL protein (a
monoclonal antibody) in the same base formulation as
described above. Vials (10 cc) were filled with 4 mL
of the above solutions and frozen by methods as
described below, and reported previously (1).
The following procedure was used to prepare vials
under the “normal” freezing conditions:
1. Samples were equilibrated on the lyophilizer shelf
at 5 °C for several hours
2. The shelf temperature was ramped linearly from 5
to ⫺50 °C at 0.3 °C/min
3. The shelf temperature was held at ⫺50 °C for at
least 5 h
This cooling strategy resulted in vials that were su-
percooled to between ⫺15 to ⫺25 °C prior to sponta-
neous ice nucleation. Our estimates were based on the
shelf temperature and visual observation of the sam-
ples during the freezing ramp.
To produce directional ice growth, samples were pre-
pared as follows:
1. Samples were equilibrated under wet ice
2. The lyophilizer shelf temperature was ramped from
5 to ⫺50 °C at 0.3 °C/min
3. Once the shelf temperature reached less than 0 °C,
each sample from step 1 was briefly contacted on
the bottom of the vial with a piece of dry ice to
induce ice nucleation then immediately placed on
the lyophilizer shelf
4. Upon completion of the ramp, the shelf temperature
was held at ⫺50 °C for at least 5 h
To produce rapidly frozen ice growth, samples were
prepared as follows
1. Samples were equilibrated under wet ice
426
2. The samples were immersed in a slurry of dry
ice– ethanol until completely frozen
3. Each sample was then placed on the lyophilizer
shelf that was precooled to ⫺50 °C
4. The shelf temperature was then held at ⫺50 °C for
at least 5 h
These cooling strategies, although impractical for
manufacturing, demonstrate the ability of the embed-
ding and imaging method to resolve finer structures
found in some cakes.
Once frozen all the samples were dried under the
following conditions:
1. The chamber pressure was set to 150 ␮m Hg pres-
sure absolute
2. The shelf temperature was ramped linearly from
⫺50 to 20 °C at 0.25 °C/min
3. The self temperature was held at 20 °C for 47 h
(this is the combined primary and the secondary
drying steps)
Upon completion of the drying, the vials were stop-
pered under vacuum and stored at ambient tempera-
ture.
Encapsulation
We used two different methods for encapsulating ly-
ophilized cakes in Dow Corning Sylgard 184 polydi-
methylsiloxane (PDMS) polymer, as discussed below.
The polymer, received as a two-component kit com-
prised of a base elastomer and a curing agent, was
prepared according to the manufacturer’s instructions.
After mixing, repeated cycling between 10 –20 kPa
absolute pressure and ambient pressure greatly facili-
tated the removal of entrained and dissolved air from
the PDMS. The mixture has a working time of several
hours (⬎2 h according to manufacturer documenta-
tion), providing ample time to perform all subsequent
operations.
During the encapsulation procedure, each sample vial
must be uncapped and briefly exposed to ambient
conditions. To minimize the possibility of excessive
moisture pickup by the lyophilized cake, a gentle dry
gas purge over the opened vial can be used.
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Figure 1
Encapsulation setups. a) Method 1, b) Method 2. (1)
modified 50 mL Eppendorf Combitip, (2) parafilm
plug, (3) wire retaining bracket, (4) 50 mL centri-
fuge tube funnel.
Method 1
Each vial containing a lyophilized cake for encapsu-
lation was set up as shown in Figure 1a. A modified 50
mL Eppendorf Combitip Plus pipette tip was used as
the reservoir for the PDMS. The tip of the Combitip
was trimmed down to the base leaving ⬃5 mm, and a
small Parafilm M plug was inserted in the opening.
Some notches were made in the plastic plunger of the
pipette tip to allow for venting. A retaining bracket
fashioned from wire (here copper was used) served to
prevent the lyophilized cake from floating atop the
PDMS once the polymer is introduced in the vial. The
wire bracket and the Combitip funnel were secured to
the vial using adhesive tape. Sufficient amount of
PDMS was poured into the funnel, and the plunger
was set in place. The assembly was placed inside a
lyophilizer and the pressure was lowered to ⬃5 kPa
absolute; the shelf temperature remained at ambient.
At this pressure additional degassing of the PDMS
occurred but most of the bubbles dissipated reasonably
Vol. 65, No. 4, July–August 2011
rapidly. The lyophilizer shelves were then partially
collapsed, just enough as to sufficiently push the
plunger down and dislodge the Parafilm plug. The
PDMS was allowed to flow from the funnel and flood
the vial for up to 1 h. Subsequently, the lyophilizer
chamber was brought back to ambient pressure, forc-
ing the polymer into the lyophilized cake interstitial
space. The vial assembly was unloaded from the ly-
ophilizer, and the funnel and wire bracket were re-
moved. The vial was then placed into an oven set at
35– 40 °C for 3– 4 days to allow the PDMS to fully
cure. Note that the PDMS can be cured at still lower
temperatures but the curing time will be lengthened.
Method 2
The setup for this method is simpler than for Method
1 in that the PDMS is introduced into the vial prior to
the application of vacuum. However, upon reducing
the pressure, substantial amount of foaming occurs.
Therefore, a funnel attached to the vial opening is
needed to prevent the PDMS from spilling out. Figure
1b shows a vial assembly ready to be filled with the
polymer. In this case, the funnel comprises a section of
a 50 mL centrifuge tube carefully attached to the vial
mouth using adhesive tape so as to prevent the PDMS
from leaking out. A retaining bracket for the lyophi-
lized cake must also be in place before introduction of
the polymer.
To encapsulate a lyophilized cake, an appropriate
amount of PDMS was poured into the funnel and the
assembly was transferred to a lyophilizer (a vacuum
oven can also be used in lieu of a lyophilizer). The
chamber pressure was then reduced to ⬃5 kPa abso-
lute. Depending on the size of the cake, more than 1 h
may be required for the foam to collapse sufficiently
before the pressure can be restored to ambient condi-
tions. Once the PDMS fully penetrated the cake, the
vial assembly was unloaded from the lyophilizer and
the funnel and wire bracket removed. The vial was
then placed into an oven set at 35– 40 °C for 3– 4 days
to allow the PDMS to fully cure.
Extraction of the Embedded Lyophilized Cake
The embedded cake was extracted by placing the vial
in a plastic bag and a vise was used to carefully shatter
it. Methanol or isopropanol can be used as wetting
agent to facilitate the removal of adhered glass frag-
ments from the PDMS plug.
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Visualization

The embedded cake that was released from the vial

was sectioned by cutting it with a sharp blade. Lon-
gitudinal and cross-sectional cuts were made and im-
aged under white illumination or illumination to en-
hance fluorescence. For the latter, a fluorescent dye
was applied from a felt tip marker (Sharpie Accent
Liquid Highlighter, orange color). The water-based
dye did not adhere to the hydrophobic polymer, and
excess dye was easily wiped off with a clean cloth.

For fluorescence imaging, the sample was lighted with

a Prolight High Power 1 watt UV LED (UV-1WS, 400
nm) equipped with a 15° focusing lens and powered by
a 350 mA Buckpuck LUXDRIVE (03021-DE-350)
LED driver. A Hoya B-390 blue filter was added to the
front of the LED focusing lens to minimize higher
wavelength light. Photographs were taken through a
Leica MZ16 stereo microscope equipped with a PLAN
APO 0.63⫻ objective and a Canon 5D SLR camera. A
Hoya K2 yellow filter was placed in front of the
microscope objective to further enhance contrast.
Results and Discussion
In the majority of cases, either proposed encapsulation
method can be used successfully. Method 1 may re-
duce the potential for entrapping bubbles, particularly
with larger cakes because vacuum is applied prior to
the introduction of the polymer. If the cake volume is
more than half the vial volume, then Method 2 should
be used to prevent overfilling the vial before the
polymer can penetrate the cake.
While the PDMS has relatively high pre-cure viscosity
(3900 cP), it exhibits very low surface tension. In most
instances, full penetration and “wetting” of lyophi-
lized cake has not been an issue in our experience. The
polymer cures into a hydrophobic, crystal-clear, me-
dium-soft material (Shore A durometer value of 48)
with excellent dimensional stability.
Figures 1– 4 are photographs of samples that have
been encapsulated and sectioned both vertically and
horizontally at about mid-section. The images, usually
taken a few minutes after sectioning the cakes, dem-
onstrate the resolving power of this technique, and the
advantage of using fluorescent staining is dramatically
apparent. Note that once a sample has been sectioned
and stained, the lyophilizate will melt. However, no
loss of optical resolution occurs because the lighting
Figure 2
Encapsulated cake containing 25 mg/mL protein
from normal freezing regiment, liquid was sub-
jected to supercooling and ice nucleated between
ⴚ15 and ⴚ25 °C. a– c) vertical section, d–f) hori-
zontal section. Frames a and d are sections photo-
graphed under white light before dye staining; all
others are from UV lighting post-staining. White
outline in frames b and e indicate magnified areas
shown in frames c and f.
highlights the edges of all cake features that were
imprinted in the PDMS. It is also possible to see
features below the exposed surface because the poly-
mer is clear and the fluorescent dye tends to penetrate
some distance into the lyophilizate.
Figure 2 shows photographs of a protein containing
sample frozen under normal condition (cooled from 5
to ⫺50 °C at 0.3 °C/min) that resulted in a cake that
exhibits sponge-like morphology. This is the expected
outcome for liquid vials that have undergone super-
cooling (ice nucleation occurred between ⫺15 to
⫺25 °C) and are subsequently frozen rapidly (1, 2). In
this instance, some cake shrinkage and cracking also
occurred during the drying. The spatial relationship
between the various parts of the cake has been cap-
tured by the PDMS, and the internal fractures are
easily observable. Some liquid phase separation may
also have occurred in this sample as evidenced by the
larger and less dense cell structure near the upper
portion of the cake (3). This latter feature would have

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Figure 3
Encapsulated cake containing 25 mg/mL protein
from directional freezing regiment; liquid was
equilibrated on wet ice and ice nucleation was in-
duced by briefly touching a piece of dry ice to the
vial bottom. The vial was then placed on back the
lyophilizer shelf at slightly <0 °C. a– c) vertical
section, d–f) horizontal section. Frames a and d are
sections photographed under white light before dye
staining; all others are from UV lighting post-stain-
ing. White outline in frames b and e indicate mag-
nified areas shown in frames c and f.
been very difficult detect without the support of the
cake structure by PDMS.
The protein-containing sample shown in Figure 3 un-
derwent directional freezing from bottom up, resulting
in a final lyophilized cake that exhibits large lamellar
structures. This morphology is consistent with a solu-
tion where ice nucleation occurred close to the freez-
ing point and ice growth proceeded relatively slowly
such as to form large ice crystals (1, 2). The voids seen
in areas near the vial wall may be localized cake
fractures due to moderate cake shrinkage.
Figure 4 shows a non-protein sample that underwent
rapid directional freezing from the bottom and sides of
the vial. This freezing regime resulted in very fine
lamellar structures oriented toward the center of the
vial. In addition, there is also evidence of cake shrink-
age causing large cracks in the periphery as well the
Vol. 65, No. 4, July–August 2011
center of the cake. This example also demonstrates the
ability to apply the fluorescence stain to samples that
do not contain protein.
Figure 5 are images of actual protein products pre-
pared in a pilot-scale lyophilizer. Figures 5a and 5b
show the vertical and horizontal cross-section, respec-
tively, of a cake that exhibits partial collapse on the
bottom half. In addition, strong neighboring vial ef-
fects are present as evidenced by the hexagonal shape
of the cake bottom. Figure 5c and 5d show the result-
ing cake structure from a vial that contained a moni-
toring thermocouple as compared with a vial without.
It is known that the presence of thermocouples tends
to induce ice nucleation at higher temperatures (lower
degree of supercooling). The sample in Figure 5c
seems to include structures formed by directional ice
crystals, consistent with slower ice crystal growth rate,
whereas that in Figure 5d exhibits a mostly sponge-
like morphology, consistent with fast ice crystal
Figure 4
Encapsulated cake without protein from direc-
tional fast freezing regiment, liquid was equili-
brated on wet ice and completely frozen in a dry
ice-ethanol bath. The vial was then placed on the
lyophilizer shelf at ⴚ50 °C. a– c) vertical section,
d–f) horizontal section. Frames a and d are sections
photographed under white light before dye stain-
ing; all others are from UV lighting post-staining.
White outline in frames b and e indicate magnified
areas shown in frames c and f.
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Figure 5
Encapsulated cakes of actual protein products pre-
pared in pilot scale lyophilizer. a) vertical cross-
section, b) horizontal cross-section take near the
bottom (approximately indicated by white line) of a
sample containing 20 mg/mL protein in 0.5 M ar-
ginine succinate. c & d) vertical cross sections of
samples containing 20 mg/mL protein formulated
with 60 mM trehalose. Sample c was sectioned as
close to the thermocouple as possible.
growth. The sample in Figure 5d was also included to
demonstrate the effect of incomplete encapsulation,
where the bright areas at the center are regions the
PDMS did not fully penetrate. These regions subse-
quently collapsed due to moisture absorption from the
water-based fluorescent dye staining after the cake
was sectioned.
In addition to the examples presented above, we have
successfully employed this technique to image a vari-
ety of lyophilized substances, including products for-
mulated with either sugars (i.e., sucrose, trehalose) or
organic salts (i.e., arginine phosphate) and of solids
contents ranging from less than 5 wt % to over 12 wt
%. Sample size ranged from a few milliliters in a 5 mL
430
vial to over 40 mL in a 100 mL vial. With care, even
highly hygroscopic material can be processed with
good results. However, very fragile lyophilized cakes
may present difficulties because they can be crushed
by the weight of the polymer when it is introduced into
the vial. The technique can easily be refined and
adapted with experimentation to suit individual re-
quirements.
Conclusion
We have presented an improved technique that is
facile and can be used to image lyophilized products
cakes without the need for specialized or expensive
equipment. We believe this to be a valuable tool for
lyophilization research and development as well as
manufacturing investigations.
Conflict of Interest Declaration
The authors declare that they have no competing in-
terests.
References
1. Patapoff, T. W.; Overcashier, D. O. The impor-
tance of freezing on lyophilization cycle develop-
ment. BioPharm 2002, 15 (3), 16 –21.
2. Searles, J. A.; Carpenter, J. F.; Randolph, T. W.
The ice nucleation temperature determines the pri-
mary drying rate of lyophilization for samples fro-
zen on a temperature-controlled shelf. J. Pharm.
Sci. 2001, 90 (7), 860 – 871.
3. Cromwell, M. E. M.; Carpenter, J. F.; Scherer, T.;
Randolph, T. W. Opalescence in Antibody Formu-
lations Is a Solution-Critical Phenomenon. In 236th
ACS National Meeting; A.O. Papers: Philadelphia,
PA, 2008.
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