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Freeze-drying of Tissues

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Musculoskeletal
TissueBanking

William W. Tomford
Director
MassachusettsGeneral Hospital Bone Bank
AssociateSurgeon
D epartmentof Ortho7aedics
M assachusetts GeneralH os7ital
AssociateProfessor
Harvard Medical School
Boston,Massachusetts

With contributionsby
JamesH. Forsell, Allen P. MacKenzie,
and D. Michael Strong

Ravenpress New York

 10036
RavenPress.Ltd', 1185 Avenueof the Americas,New York, New York

protectedby
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Made in the United Statesof America

Data
Library of CongressCataloging-in-Publication

Musculoskeletaltissuebanking/William W. Tomford'
p. cm.
Iniludes bibliographicalreferencesand index'
rsBN 0-88167-995-X
1. Musculoskeletalbanks. 2' TissueBanks' I' Tomford, William W'
IDNLM: 1. Bone Transplantation' 2' Muscles-transplantation'
3. Transplantation, Homologous. wE 190M9851
RDl28.M88 1993
362.1'783-dc20
DNLM/DLC
for Library of Congress 92-496r6
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The material containedin this volume was submittedas previouslyunpublished

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98 '76 54 321
 Freeze-Drying of Tissues
D. Michael Strong and Allen P. MacKenzie

Tissuebanking, as the name implies, requiresan ability to pre-

serve various tissues for different periods of time. This is
achievedby cold storagerangingfrom *4oC to - 196'C, aswell
as by lyophllization, or freeze-drying.
A biologic material, suchastissue,which driesfrom the liquid
state,will shrink, will becomerelatively insoluble,and may suf-
fer permanent chemical alteration. To extend the preservation
time and avoid the dehydratingeffects,tissuesare frozen to im-
mobilize the water. An extension of this processis the use of
freeze-dryingwhich, when appropriatelycontrolled, hasthe pri-
mary advantagesof preventing shrinkage,maintaining solubil-
ity, minimizing chemical changes,and facilitating storage.
This chapterwill cover the historical aspectsof freeze-drying
as it appliesto tissuebanking, explain the basic principles in-
volved,describethe equipmentrequirementsand protocolsused
for tissues,and discussfactorsthat may affecttissuequality and
quality control requirementsto validate the process.

HISTORICAL PERSPECTIVES

Although freeze-dryinghas become one of the most widely

usedmethods of preservingtissue,particularly bone, for trans-
plantation, the earliestapplicationwasfor laboratoryprocedures
for histologictechniques(5). The report by Shackell(83) in 1909
on the use of freeze-dryingfor biologic materialsled to numer-
ous investigationsinto its usefor other biologicals,suchascom-
plement, antisera,viruses,and bacteria(26).

181
182 FREEZE-DRYING

The first issuedpatent for the freeze-dryingprocesswasthat of

Elser(24) in 1934,which describedthe methodof drying frozen
material under vacuum using a solid carbon dioxide cold trap.
The one significantmissingelementfrom this disclosurewasthe
use of heat to supply the heat of sublimation. This patent was
followed closelyby a descriptionof a designfor the commercial
useof freeze-dryingusing the principle of the cold trap and pro-
viding for the rapid freezingof the samplein the samelow-tem-
peraturebath used to cool the condenser(28). The report by
Greavesand Adair (a1)in 1939provideda scientificdescription
of mechanicallyrefrigeratedequipment, including the various
parametersconcerned with vapor pressure,vacuum, relative
temperatures,and effectsof vapor flow. It was also during this
time that the useof residualmoisturedeterminationsfor quality
control of the drying processwas introduced (30).
During World War II, the developmentof freeze-dryingand
the needfor largescaleproduction ofplasma forthe treatment of
casualtiesprovided the impetus for the development of large
scaleproduction freeze-dryers(29). The work of investigators
such as Flosdorf and Greavesand the successachieved with
freeze-dryingplasmain World War II led to the extensionof this
technologyto the preservationof human bone for usein repara-
tive surgeryduring the Korean War. The U.S. Navy established
the first tissuebank under the direction of an orthopedic sur-
geon,Dr. GeorgeHyatt, whosecollaborationwith Dr. Flosdorf
resultedin the first publication of the useof freeze-dryingfor the
preservationof bone grafts(27,54),Sincethis introduction of
freeze-driedbonefor clinical use,many other freeze-driedtissues
have been usedclinically with varying degreesof success(51).
Theseincludefascia(82),dura mater (1,20),arteries(13),veins
(61),skin (81),tendons(76),and heartvalves(7).

THE PRINCIPLES OF FREEZE-DRYING

Freeze-dryingrequiresthe conversionof water to ice (or the

presenceof ice if the sampleis alreadyfrozen), the sublimation
of that ice, and the simultaneousand/or subsequent removalof
some or all of the water not converted to ice bv the freezine
 FREEZE-DRYING 183

process.The loss of ice by sublimation facilitatesthe develop-

ment of a porous structure that facilitatesthe continued subli-
mation of ice. The sameporous structurefavorsthe evaporation
and loss of the unfrozen water.
Freezing and freeze-drying are driven by thermodynamic
forcesand opposedby kinetic resistances.Materials freezewith
sufficientcooling to developan ice phasein which water mole-
cules occupy statesof the lowest availableenergy.Some water
remains unfrozen, even at very low temperatures,and is not
convertedto ice either by a slowerfreezingor by freezingfor a
longer time. In other words, the force that drives the freezing
processhas been reducedto zero.Unfrozen water can also be-
cometrappedat subfreezingtemperaturesif freezingis especially
rapid (a slower freezingwould give the samewater time to find
its way to growing ice crystals).In this situation, a driving force
survivesbut is ineffective in the face of a very high diffusive
resistance.
The general experienceof the freezing of biologic material
suggests that so-called"completely frozen" (water-soluble,pro-
tein-rich)biologicsystemsmay containasmuch as20Voto 25Vo
unfrozenwater(referred,asa percentage,to the dry weight of the
samematerial).A very rapid freezing(e.g.,with a liquid cryogen)
can increasethis value by as much as 5Vo(i.e.,to 25Voto 30Vo).
None of these figures is very large, and nothing prevents the
crystallizationofthe greaterpart of the waterfound in a represen-
tative soft tissueduring a typical freezingto -40'C or to a lower
temperature.
The sublimation of ice is driven by a vapor pressuredifference
(and is resistedin various ways).With decreasingtemperature,
the vapor pressureof ice falls smoothly and continuously to
demonstrate a roughly semilogarithmic temperature depen-
dencebestsummarizedasan approximatelythreefoldreduction
in equilibrium vapor pressureper 10'C interval. The depen-
denceis illustratedin Table 5.1.
We may chooseany two temperaturesfrom Table 5.1 and
determine the sublimation of ice during freeze-drying.Ice in a
sampletissueat -30'C will exert a vapor pressureof approxi-
mately 0.3 mmHg (or 300 pm or 0.38 mbar). Ice accumulated
184 FREEZE-DRYING

TABLE 5.1. The vapor pressureof ice as a function of temperature

Temperature Pressure
('c) mmHg mbar
0 4.579 4 ,5 7 9 4,579 6.105
-5 3 .0 1 3 3 ,0 1 3 3,013 4.O17
-10 1 .9 5 0 1 ,9 5 0 1,950 2.600
-15 '1.241 1 ,2 4 1 1,241 1.655
-20 o .7 7 6 776 776 1.035
-25 o.476 476 476 0.635
-30 0.2859 286 286 0.3812
-35 0 .1 6 8 1 168 168 o.2241
-40 0.0966 9 6 .6 96.6 0.1288
-45 0.0541 54.1 54.1 0.0721
-50 0.0296 29.6 29.6 0.0395
-55 0 .0 1 5 7 15.7 15.7 0.0209
-60 0.0081 8 .1 8.1 0.0108
-65 0.00403 4 .O 4.O 0.0054
-70 0 .0 0 1 9 1 .9 ' t.9 0.0026
-80 0.0004 o.4 o.4 0.0005
-90 0.00007 o.o7 o.o7 0.0001
-100 0.00001 0.01 0.01 0.000013

on a mechanicallyrefrigeratedcondensercoil at, for example,

-60"C will exerta vapor pressureof 0.008mmHg (0.01mbar).
Freeze-dryingwould proceedwith a net driving force of 0.278
mmHg (0.37 mbar). Theseare very small pressuredifferentials
by any practical reckoning. They can be increasedonly by rais-
ing the temperature of the ice in the sample material (i.e., by
warming the frozentissue)or by loweringthe condensertemper-
ature.The samplematerial cannot be warmed abovea tempera-
ture at which the melting processstarts.The freeze-dryingpro-
cessitself is compromised when the sample material is dried
from a lessthan fully frozenstate.Nor canthe condensertemper-
ature be loweredbelow -70"C to any greatadvantage.One sees
readily from Table 5.1 that further reductionsin condensertem-
peratureto -80"C or to -100"C effectrapidly diminishingre-
turns. Experimentally,tissuemeasuring10 mm to its centerhas
a predicteddrying time of approximately 1 year at -60'C (88).
At -75"C, the temperaturecited by Urist et al. (93) for freeze-
drying bone powder,it is predictedthat the drying time would be
 FREEZE-DRYING 185

greaterthan a decadefor tissue22 mm in depth, 4 months for 2

mm in depth,and only 3 to 4 daysfor 2 mm or less(74).
Water vapor encounters a successionof resistancesin the
courseof its passagefrom ice in the sample(typically, from the
ice in the "freeze-dryingfront") to a mechanicallyrefrigerated
condenser(whereit is reconvertedto ice). The water molecules
must find their way through a developingdry layer aboveand/or
under and/or around the ice remaining in the tissueat any mo-
ment, through the head space in any individual container,
through the flutes of a split rubber stopper and/or any sterile
filter or wrapping,through the spacesbetweenthe shelvesin the
chamber,and, lastly, through any remaining spacesdetermined
by choices related to condenserconfiguration. Typically, the
greatestofthese resistancesexistsin the "developingdry layer."
This resistancecan be lessenedby reducingthe thicknessof the
sample itself, or the fiIl volume, if the sample is a liquid or a
slurry containedin a vial or a tray. It is further minimized by a
careful choice of freezing procedure. Larger ice crystals (slow
freezing)are sublimed to furnish more porous "developing dry
layers." But the relationship between ice crystal size and dry
layer porosity is not well understoodand variesfrom one kind of
sampleto another.Other resistances to vapor transfer,most par-
ticularly those associatedwith the freeze-dryingequipment it-
self, are typically determined for the user by the freeze-drier
manufacturer. Theselatter resistanceshave been reducedby a
continuing manufacturing experience.They cannot be lowered
further without additional cost (which will not generally be
justified).
Freeze-dryinginvolvesthe transferof very considerablequan-
tities of heat without which the transfer of water vapor would
slow and stop. The sublimation of ice is accompaniedby the
absorption, at the subliming ice interface (or "freeze-drying
front"), of 675 caloriesper gram of ice sublimed.This heat must
come from adjacent sources.If the heat is not provided, the
sample material will cool by evaporation. The temperature of
the ice in the samplewill drop, and the driving force for vapor
transferwill be diminished. Were the samplematerial to be per-
fectly thermally insulated, it could (theoretically) cool to the
186 FREEZE-DRYING

temperatureof the ice on the condenser.Then freeze-drying

would stop altogether.
Trapping the water vapor arriving at the condenserrequires
the removalof the same675 caloriesper gram of ice developed.
If it is not removed,the temperatureof the ice on the condenser
will rise. Table 5.1 predictsthe developmentof back-pressure
that, once again,reducesthe driving force for vapor transferbut
for a differentreason.Ifcondenserrefrigerationceasedand the
accumulatedice wereperfectlythermally insulated,the tempera-
ture of the ice would rise until it equaled that of the sample
material.
It will be clearat this point that freeze-dryingwill require very
careful control. Too little heat and the sampletemperaturewill
fall; the freeze-dryingrate will fall correspondingly.Too much
heat and the sampletemperaturewill rise. Freeze-dryingrates
may rise but only with the warming of the freeze-dryingfront
above some maximum safe value. Too powerful a condenser
and the watervapormay be collectedtoo efficiently;thepressure
of the water vapor in the chamberwith the samplematerial may
then fall to such a low value that heat no longer flows readily
from shelvesto product. Freeze-dryingslowsaccordingly.A too-
small condenserwill warm under continued load (although it
may continue to function) and causefreeze-dryingto proceedat
a highertemperature.It will cool againasthe load is reduced(but
will havecompromisedgood control).
Numerous efforts have been made to effect both heat and
water vapor transfer during freeze-dryingand to do it in such a
way that sampletemperatures(a) do not fall so low that driving
forcesare reducedand (b) do not rise abovethe maximum safe
temperaturesfor the particular products. Gas pressurecontrol
hasemergedasthe singlemost promising method and deservesa
brief description.More than one variationofthe methodis now
in use.
Typically, meansare usedto maintain gaspressuresat values
in the rangeof 0. 1 mmHg (0.13mbar) to 0.3 mmHg (0.4 mbar)
during the time required to sublime ice. Gas pressurecontrol
may be achievedby (a) the intermittent closureof the connec-
tion betweenthe condenserand the mechanicalvacuum pump;
 FREEZE-DRYING 187

(b) the "throttling" of the flow of water vapor to the condenser;

or (c) the "bleeding" of pure dry nitrogen gas into the freeze-
dryer. All of the aforementionedprocedureswork more or less
equally well if they facilitate the flow of heat from the shelvesto
the product (whereheat transferwould be reducedat lower sys-
tem pressures).Controlled chamber pressuresin many fteeze-
drying operationshave been arrived at by trial and error that
could have been avoided had the maximum safefreeze-drying
temperaturealreadybeenknown. Table 5.1 providesthe vapor
pressuresof ice at varioustemperaturesand suggests alsothat the
sublimation of ice will not proceed at a useful tate at a particular
temperatureuntil the system pressure is reduced below the value
in question. Knowing a maximum safe temperature above
which an ice interfaceshould not warm during the sublimation
process,we may consult Table 5.1 (or an extendedversion
thereof) and define a maximum gaspressurefor pressurecon-
trol. A generaloperatingexperiencetestifiesto the utility of the
approach.
A considerableeffort is often made to follow the course of
primary drying (i.e., the sublimation of ice) and to test for its
completion. Primary drying can be followed readily where a
freeze-dryingfront can be seento move through a samplemate-
rial, as it can when an aqueoussolution makesperfect contact
with a glasssurface.Tissuebankersare typically lessfortunate.
Freeze-dryingfronts are developedat the free surfacesof less
than regularobjectsand are(almostcertainly)themselvesirregu-
lar, although they cannot be seen directly. The progressof a
freeze-dryingfront is frequently a matter for speculationin such
a circumstance.
Good freeze-dryingpractice has generallydictated the use of
thermocoupleprobes or other similar sensorsin samplemate-
rials.Probesareplacedat the bottom centerof simplecylindrical
samplevolumes(e.g.,in pharmaceuticalsolutionsin glassvials)
on the assumptionthat the freeze-dryingfront will descenduni-
formly through the samplevolume. Thermocoupleprobesdem-
onstratethe evaporativecooling of the sampleassociatedwith
the sublimation of ice. The absenceof any evaporativecooling is
generallytaken to indicatethe completion of primary drying but
 18 8 FREEZE-DRYING

depends,ofcourse, on the advantageousor "correct" placement

of the thermocouples.
An alternativeprocedure,known variouslyas"the barometric
method" and the "pressure-risetechnique," overcomesdifficul-
ties inherent in the useof thermocouplesor other probesduring
primary drying. Ice demonstratesan equilibrium vapor pressure
that dependsvery strongly on temperature,as we have said (see
Fig. 5.1 and Table 5.1) and can be put to work, in a way, to
measure its own temperature and to document its own
disappearance.
Freeze-dryingis begunin the usualway and is continued,with

103- -100 -80

1 03

102-

tnl
o) 10'
I
E
E too-
1 00
E
f
q
o.
=
o
E 10- ' : o
o 1o-1
b o
9 1o*.
c)
1o{
3 G
=
10-:
1 03

1o1-
10-4,

1o5- 1o'5
-100 -80 -60 -40 -20 0 20 40 60 80 100
("C)
Temperature
FlG. 5.1. A part of the pressure-temperature diagramfor pure water. Vapor
pressuresof ice and liquidwater are representedas functionsof tempera-
turesfrom -100"C to 0"C and from 0'C to 100"C,respectively. The melting
point of pure ice is shown as a functionof hydrostaticpressure.The "triple
point" representsthe stablecoexistenceof ice,liquidwater,and water vapor
in the absenceof air.
 FREEZE-DRYING 189

or without pressurecontrol, until, at a predeterminedmoment,

the chamberwith the samplematerial is abruptly isolatedfrom
the condenserto which water vapor was smoothly flowing. Left
with nowhereto go, water vapor accumulatesin the presenceof
the freeze-dryingsample material. Operating experiencesug-
geststhat water vapor pressuresrise to equilibrium values in
severalsecondsin tlpical casesand that freeze-dryingis thus
intemrpted. The riseto an equilibrium pressurecan be followed
and the pressuremeasured,after which the samplematerial can
be reconnectedto the condenser(by way of the opening of the
requisitevalve).The freeze-dryingis thus allowedto resumeand
to continue.
Sampletemperaturesare derivedfrom pressuresto which iso-
lated samplechambersrise in accordancewith Table 5'1. Were
the pressureto rise, for example, to a value of approximately
0.475mmHg (0.635mbar)aftertheisolationofthe samplemate-
rial (and not to rise further), the readingwould (from Table 5.1)
indicate a freeze-dryingfront temperatureof -25"C. The ice in
the samplematerial serves,therefore,as its own thermometer,
but the pressurereadingdoesnot, of itself, revealhow much ice
remains to be sublimed (or, for that matter, where it is to
be found).
Consecutivepressure-risetestsconductedat (e.g.,hourly) in-
tervals usually demonstrategradually increasingfreeze-drying
front temperaturesuntil, quite suddenly,pressuresfail to rise to
respectiveanticipated values. These rather sudden changesin
pressure-risebehavior are correlatedwith the disappearanceof
the last ice. Thus, the barometric or pressure-risemethod signals
the completion of so-called primary drying.
The barometric method proves generally to work well but
requires a certain care and understanding on the part of the
operator.Freeze-dryersshould not leak, or the method will not
work. Vacuum gaugesshould be calibrated for water vapor
(most of the lessexpensivegaugesare calibrateddifferently for
different gases),or sample temperatureswill be in error. Gas
bleedsto the chambershould be turned offbefore any pressure-
rise measurements(but this can be done automatically).Sample
material isolatedfrom an adjacentcondenserwill tend to warm
192 FREEZE-DRYING

samplematerial to the condenser.Freeze-dryermanufacturers

sizevacuum pumps to allow the evacuationof the freeze-dryer
to, say,0.1 mmHg (or 100pm) in l5 or 20 minutes,afterwhich
the pump is run largely to remove air "outgassed"from equip-
ment and product and, of course,to pump inert gas (e.g.,dry
nitrogen) "bled" into the equipment to control chamber pres-
sure. Mechanical vacuum pumps accumulate solventssuch as
ethanol, and their effectivenessis compromised accordingly.
Vacuum pumping systemsmust incorporate cryogenictraps to
protect mechanical pumps from any organic solvent vapors
wherethesesubstancesare present.
The pressurein the freeze-dryeris measuredby one or more of
several sorts of vacuum gauges.Thermocouple and Pirani
gaugeshave proved to be economic and effective,but their cali-
bration dependsin each caseon the gas presentin the fteeze-
dryer. Capacitancemanometer gaugesread correctly regardless
of the nature of the gasbut are more expensive.Mercury ma-
nometers (Mcleod gaugesincluded) were used in the past to
measurepressurein somefreeze-dryers(much asthey wereused
in many laboratory vacuum applications)but should neverbe
usedin tissuebanking.Mercury demonstratesa smallbut signifi-
cant vapor pressureat room temperature and can be distilled
from the gauge(into the freeze-dryer)with very little effort. The
smallesttrace of mercury would be causefor alarm.
Freeze-dryersare, ever more frequently, equipped with pro-
grammable controllers that allow an operator to predetermine
the shelftemperatureand, in many cases,the chamberpressure
asa function of time (i.e.,to write a "freeze-dryingcycle") and to
accomplishan entire freeze-dryingat the touch of a button. The
simplercontrol systemsimposefreeze-dryingconditionson sam-
ple material that may or may not freeze-dryaccordingto plan.
More advancedcontrol systemsreceiveinformation from the
samplematerial (e.g.,by way ofthermocouplesignalorbaromet-
ric test)and may adjust shelftemperatureandlor chamberpres-
sure to maintain a required sampletemperature.Control sys-
tems may incorporate many safeguardsdesigned to protect
sample material, insofar as this is possible,during subsystem
failure. A control systemmay likewise protect the equipment
 FREEZE-DRYING 193

(and, as much as possible,material being freeze-dried)during

power failures(e.g.,when line voltagedrops too low or the flow
of cooling water is intemrpted).
Freeze-dryersmay be designedand constructedfor steiliza-
tion in one of a number of ways.Ethylene oxide sterilizationis,
perhaps,accomplishedmost conveniently;it requiresthe attach-
ment of a systemof inlets and outlets,suitably valved,and con-
nectionsand interlocks to protect laboratory personnel.Essen-
tially any laboratory or small-scaleproduction freeze-dryercan
be adaptedto ethyleneoxide sterilization.Unfortunately, many
concerns surround the use of ethylene oxide in freeze-dryers
usedin tissuebanking.
Freeze-dryersmay be sterilizedwith pure steambut must be
designedand constructedspeciallyforthe purpose.Freeze-dryer
chambersand condensersarenormally constructedto withstand
an external pressureof a full atmosphere(they are, after all,
operatedunder an essentiallyfull vacuum), but they must also
be constructedto withstand an excessinternal pressureof an
atmosphereor more if they are to be steam sterilizable(they
must, that is to say,be constructedmuch like an autoclave).It is
not surprisingthat steam-sterilizablefreeze-dryingequipment is
very expensive.
Other methodsof sterilizingfreeze-dryersare currently under
development,most notably methods that might use hydrogen
peroxide.Long known for its sterilantproperties,hydrogenper-
oxide seemsto be suited to the sterilization of freeze-drying
equipment.Tissuebankerswould almost certainly benefit from
a method that offeredan alternativeto ethyleneoxide steriliza-
tion and did not require the use of pressure-ratedequipment.

THE EFFECTS OF FREEZE.DRYING ON TISSUES

As previouslymentioned,a gooddealofresearchon the useof

freeze-dryingfor bone and soft tissuepreservationwas carried
out during the two decadesafter the establishmentof the Navy
TissueBank(71,74). During this period, this preservationtech-
nologybecameroutinely usedfor clinical applications.Unfortu-
natelv. unlike the researchconducted on the effectsof freeze-
194 FREEZE-DRYING

drying on viruses(45) and other biologic compounds,which

usedobjectivemeasurementsto definethe variousparametersof
the freeze-dryingprocess(39), most of the reportson tissuehave
been primarily clinical. Thus, most of these publications give
little information on the preciseproceduresusedin the lyophili-
zation process;therefore, the varying clinical results obtained
cannot be relatedto the differencesin techniques.
The primary concern of the tissuebanker is to preservethe
function of the graft in question. For example,bone has three
primary uses:structure, osteoconduction,and osteoinduction.
The factors that affect the physical characteristicsof the graft
may or may not haveany relationshipto the biologic properties.
Thus, a protocol designedto maintain the biomechanical
strength of an intact cortical shaft may be different from one
designedto preservethe osteoinductiveproteinsof bonepowder.
Thereare,however,certainprocessingstepsthat can havedelete-
rious effectson both structure and function.
Processingchemicalssuch as ethanol, if not completely re-
moved from the graft, will not freezeat the temperatureusedin
primary drying and thus may result in tissuethat is not truly
freeze-dried.In addition, the alcohol left in the tissuewill vapor-
ize and generatea condition known as outgassing,resulting in
difficulties for the equipment to maintain or reach adequate
vacuum. Similarly, if tissueis sterilizedwith ethyleneoxide and
residualsof ethyleneglycol and ethylenechlorohydrin remain,
thesecompoundswill also resistfreezingand be more concen-
trated by the processof water vapor removal. Suchresidualscan
resultin significantclinical reactions,including anaphylaxis(75)
and tissuedestruction(53).
Other sterilization proceduresin combination with freeze-
drying may have additive effects.Early studies demonstrated
that irradiation sterilization of freeze-driedbone resulted in
graftsthat were not histologicallyacceptedaswell as were those
that were only freeze-driedbeforetransplantation(50). This ob-
servationhas been confirmed by others in comparing the me-
chanicalstrengthof grafts (10,55).However,this effectis not
seenif the order is reversed(i.e., if irradiation is followed by
freeze-drying) (47,49,77). In addition, irradiation sterilization is
 FREEZE-DRYING 195

significantly more effective if more water is available for the

generationoffree oxygenradicals(12).
A discussionof the effectsof freeze-dryingmust include the
issueof freezing,sincethe former cannot occur without the lat-
ter. The rate of freezing,the composition of the tissue,the water
content,and the presenceof crystallineand noncrystallineresid-
ualsare all factorsthat affectice crystalsizeand shapeand influ-
encethe sublimationprocess(60).For example,a slow cooling
resultsin larger ice crystalsthat leave a more open spaceafter
sublimation and providesthe ability for more rapid removal of
water vapor. Slowfreezingis particularly important in the recov-
ery of living microorganismsafter sublimation (40). For bone, a
nonviable tissue, torsional strength remains unchanged after
freezingto temperaturesof -20"C to -196'C. However,com-
pressionloadingstrengthcanbe reducedby lUvoto 20Vo( 55,73).
Freezingdoesnot seemto have a significantbiomechanicalef-
fecton tendons(52) or fascia(48).Nevertheless, the efficiencyof
drying from the frozen stateis dependentupon satisfactorysub-
limation of water from ice at all tissuedepths.If the frozen state
is not maintained during this processbecauseof too much heat
being applied to the frozen boundary or inadequate freeztng
(e.g.,ethanol, ethyleneglycol), there can be a collapseof the
peripheral channels with entrapment of water and solute
(58,59).Theseeffectscould explainthe differences in the results
of biomechanicaltestingof bone.Suchreportsincludeincreases
(73) or no changesin compressivestrength(10,15)and signifi-
cant reductionsin bendingstrength(92). Crackingof bone has
also been describedafter rehydration(72),but whetherthis is
due to the effectsof a too rapid freezingin liquid nitrogen,which
hasbeen seenin tissuesuch as heart valves(4), or to freeze-dry
melting and the resultingcollapse,as might be seenin air-dried
bone,remainsto be determined.
Another variable that has not been well defined in many of
thesestudiesis the rehydration procedureused.It hasbeendem-
onstratedthat rehydration outside of the evacuatedcontainer
(ex vacuo)for as much as 24 hours may be inadequate(10).
Recent studieshave shown that in vacuo rehydration is much
more efficient than ex yacuorehvdration for both cortical (25)
196 FREEZE-DRYING

and cancellousbone (68). In addition, the solution usedfor re-

hydration hasbeenshownto be important in retention of activ-
ity. The use of 5Vodextran,for example,is optimal in reconsti-
tuting dried mitochondria (44). Studies of this type have not
been carried out for tissue.
Just asimproper rehydration techniquescan affectthe biome-
chanicalstrengthof freeze-driedbone, so too can excessivedry-
ing, particularly secondary drying. Greiff and Rightse[ (a6)
showedthat freeze-dryingof influenza virus to residual mois-
tures of lessthan 17oresulted in poor recoveries.Injury from
drying can be causedby chemical denaturation due to concen-
tration effectsof solutes(aspreviouslymentioned),recrystalliza-
tion of saltsor hydratesformed from eutecticsolutions (58), or
removal of bound water during secondarydrying, resulting in
irreversibledenaturation or cross-linkingof moleculessuch as
collagen(10).
Sucheffectscan occur at the time of freeze-dryingor,particu-
larly in the caseof the first two, during storage.For this reason,
the exclusionof oxygenfrom containersof dried material is of
prime importance. It has been reported that stabilitiesof dried
suspensionsof virus are increasedby storageunder gasessuchas
helium and hydrogen(47).In addition to the possibility of over-
drying, high residual moisture levels and permeablepackaging
may result in ever-increasingdegradationof the product. Thus,
the use ofrigid containerssuchas glassprovidesgreaterprotec-
tion and longer stability, assumingvacuum integrity.
The storagetemperatureof the dried product is another vari-
ablethat will determinethe shelflife of a given material.Stability
studiesof viruseshave shown that lower storagetemperatures
result in prolongation of storagellfe (44,45).Thesestudieshave
been extendedto predict the stability of viruses and proteins
using increasingtemperaturesfrom 4"C to 37"C to determine
shelflife. Sinceincreasingtemperatureresultsin acceleratedloss
of activity, Arrhenius plots can predict storagestability (43).
Although such studieshave not beencarried out for tissue,they
have becomea standardin the pharmaceuticalindustry for de-
termining expiration datesfor numerous biologicals.Although
this processis most likely to apply to the biologic activity of
 FREEZE-DRYING 197

bone, it may not be as relevant for the structural components

ofbone.
Despite the reports of deleteriouseffectsof freeze-dryingon
variousgrafts,this procedurehasmany advantages.The success-
ful cliniial useof freeze-driedtissuesfor a variety of applications
has been widely reported and has included fascialata ( 11,91),
tendon(18,19,52,70), dura mater( 1), skin (3,81), cardiovascular
tissue ( 13),andbone (2 6,57,62-64,66,69,7
1,22,48,50,5 8-80,85,86)'
In addition to the benefitsofstorage,changes in biologic activ-
ity can be beneficial.Severalstudieson the reduced immunoge-
nicity of bone as a result of freeze-dryinghave been reported
(16,17).Thesestudieswere done in a variety of animal systems
using a variety of immunologic techniquesto determinesensiti-
zatiin(32-34). Sensitizationin thesesystemsseemsto be due to
the cellularcomponentsofthe grafts;thus,the reduction ofthese
elementsby processingsteps,freezing,or freeze-dryingresultsin
a reducedantigentoad (3 1,89).This effecthasalsobeenreported
for other freeie-dried tissuessuch as skin (2). Studieson both
humeral and cellular responsesin recipients of human freeze-
dried skin allograftshaveconfirmed the lack of immunogenicity
(35,36).In contrast,theredoesseemto be a limited responseto
histocompatibility antigens in some recipients of freeze-dried
bone grafts(32). In animal studies,the loss of immunogenicity
,."-, to result in a definite long-term survival advantagefor
lyophilized veins over cryopreservedgraftsusedasvascularsub-
stitutes(37,38).
The reduction in cellular material in freeze-driedgrafts can
alsoreducethe risk of infection. Although freeze-dryinghasbeen
routinely used for the preservationof virusesand bacteria,the
parametersfor optimal recoverymust be followed,including the
useof preservativesolutions(44). Preservativesolutionsare not
routinily used for protecting tissuesfrom freeze-dryrnginjury,
so resident infectious organismswill be adverselyaffected.A
recentdemonstrationof this is the report of the transplantation
of tissue from a patient with acquired immunodeficiency dis-
ease.Recipientsof frozen tissuefrom this donor were shown to
be antibody-positive to the human immunodeficiency virus,
whereasthosepatientswho receivedfreeze-driedtissuewereneg-
19 8 FREEZE-DRYING

ative (84). This effectis partially due to the fact that the donor
had beeninfected shortly beforedeath (HIV antibody-negative,
antigen-positive)and thus had a low viral burden. Second,HIV
is more susceptibleto drying and other disinfectantsthan are
other viruses(90). HIV hasbeenshown to survivefreeze-drying
if presentin higherconcentrations,asin tissuesfrom donorswho
havedied of AIDS (14).

QUALITY CONTROL

Oncethe freezingand freeze-dryingcharacteristicsof a partic-

ular tissueare known, the processcan be monitored and con-
trolled. In addition to having standardproceduresdescribingthe
operation of the equipment according to the manufacturer's
guidelinesand instituting the routine maintenancethat must be
performed to assureoptimal operation, monitoring should be
conductedthroughout the freeze-dryingcycle.
The AATB TechnicalManual (6) statesthefollowingminimal
requirementsfor a quality control program.
(A) A quality control program for monitoring the performanceof the
freeze-dryershall be documented.This documentationshall include, as
a minimum, demonstrationthat the freeze-dryerhas the capability of
holding the shelftemperaturewithin acceptablelimits and that the sys-
tem can achievea vacuum adequatefor freeze-drying.
(B) Each freeze-dryingcycle shall be monitored during operation for
shelftemperature,condensertemperatureand vacuum and the values
recordedat least every 24 hours. After a standardizedprocedure for
freeze-dryinghas been developed,each freeze-dryingcycle must be
clearlydocumented.Documentationfrom eachfreeze-dryingcycleshall
include: method/length sterilization, and length/temperature/vacuum
pressureat eachstepofthe cycle.

Ideally, the freeze-dryershould have a recording device to

allow measurement ofthe shelftemperature,the condensertem-
perature, the product temperature,and the chamber pressure.
Becauseof the varied contentsof any freeze-dryerrun (i.e.,corti-
cal or cancellous,size variations,etc.), chart recordingsmay
showvariances.Thus, eachcyclemust be individually evaluated
to determinea possibleexplanation of variances.When chemi-
cals such as ethyleneoxide and ethanol are usedin processing,
pressuredifferenceswill be seen.
 FREEZE-DRYING 199

In tissuebanking,the most common method for monitoring

the primary drying phaseis by meansof a temperatureprobe in
the tissue.Although this is acceptablepractice,it is only practica'
ble as long as the probe is completely surrounded by frozen
material. Since the tissue surrounding the probe often dries
fasterthan the remaining tissue,the temperaturesmeasuredare
usually not very accurate.As mentioned previously, the most
reliable and precisetechniquefor measuringthe sublimation of
ice, during either primary or secondarydrying, is by measuring
the saturationvapor pressureafter the drying chamberhasbeen
isolated for a few secondsfrom the condenser.Unfortunately,
not all freeze-dryingequipment is capableof this function. Dur-
ing the freeze-dryingprocess,aslong asice is still subliming and
being transformed to water vapor, pressure in the drying
chamber will rise when the valve betweenthe drying chamber
and condenseris closed.If this pressuferiseis minimal or if there
is no pressureincreaseat all within a few minutes, either second-
ary drying may be startedor, dependingon the tissue,the freeze-
drying cyclecan be terminated.This method is very accurateif a
drying chamberis vacuum tight.
Optimal freeze-drying is dependent on a vacuum-tight
chamber.Therefore,it is recommendedthat the systemevacua-
tion rate and leak rate be determinedroutinely on freeze-dryers
(94). Other equipment checks that should be routinely per-
formed include regular oil changesor routine observationfor
contamination of oil with water, indicated by a cloudy appear-
ance of the oil. In addition, chemicalsused during processing
steps,such as ethanol and ethyleneoxide, which may contami-
nate the water vapor, should be monitored by routine review of
vapor pressures.
Many tissuebanks freeze-drytissuefrom multiple donorsin a
singlerun. Therefore,controls needto be put in placeto assure
that cross-contaminationfrom one donor to another has not
occurred. It is therefore recommended that only culture-
negativetissuebe freeze-driedand that the chamber and con-
denser be verified as sterile before freeze-drying,since viable
microorganismscan be transferredby water vapor from vial to
vial dependingon the configuration of the drying chamber,the
200 FREEZE-DRYING

tissues,and the condenseroutlet (8,9,87).In addition to the

microbiologic testing done on the sterilized chamber before
freeze-drying,a representativesampling of bottles of tissue
(usually 10%) should also be tested. All results and records
should be recordedfor eachrun.
Methods of chamber sterilization/disinfectionhave included
chemicals,as in gassterilization with ETO or formaldehydeor
simple alcohol wiping of the shelves.Chemical sterilization
raisesconcernabout contamination of the product and the effect
on vapor pressureduring freeze-drying.Commercially, steam
sterilization is the method of choice; however, this requires
much more expensiveequipment, since the chamber must be
ableto withstand high pressures.Of particular interestis the use
of hydrogenperoxideand ozone,currently under development.
Preliminary resultsin this laboratory suggestthat small volumes
of 30Voaqueoushydrogenperoxide solutions can be vaporized
under controlled conditions to provide effectivesterilization of
the freeze-dryerchamberin a relatively short time.
The use of residual moisture determinations on freeze-dried
tissueshas become a routine quality control step for all tissue
banks.The variousmethodsfor measuringresidualmoisture, as
well as the various drawbacksfor each of thesemethods, have
been well reviewed (65). As previously mentioned, a certain
amount of bound water will remain in tissue after freeze-drying
regardlessofthe lengthofthe process.The AATB standardsstate
that the residualmoisture of tissueafter fueeze-dryingshould be
lessthan 5Vo,?smeasuredby the gravimetrictechnique,or 8Vo,ds
measuredby magnetic resonance.The initial water content in
bone varies from 20Vofor cortical bone to over 30Vofor cancel-
lous bone. Thus, if cortical and cancellousbone are freeze-dried
in the same freeze-dryrngrun, the amount of water to be re-
moved is significantlydifferent and the rate of removal will also
be different (65). Therefore,it is important that representative
samplesof eachtype of tissuebe included in the freeze-dryerrun
to be usedfor residualmoisture determinations.In addition, the
drying rate and final moisture content differ from sample to
sample,dependingon the placement of the samplewithin the
chamber, with faster drying rates at the periphery (42).Thus,
 FREEZE-DRYING 201

samplesused for residual moisture determination should be

placedto achievethe best possiblerepresentation.
Residualmoisture determinationscan vary significantlyfrom
run to run and should be standardized(23,67).The gravimetric
method, usedby most tissuebanks,hasbeenreportedto have a
standarddeviation of 3.6Voto 9.lVo (67). Recently,a quality
control exercisewas carried out by the WesternAssociationof
TissueBanks to comparethe resultsof residualmoisture deter-
minations among banks. Freeze-driedcortical and cancellous
bone sampleswere submittedto a singlebank for residualmois-
ture determinations using a gravimetric standard.Drying was
carried out in a 90'C vacuum oven, and weightswere recorded
daily until two consecutiveweighingsshowedno lossof weight.
The individual banks measuredresidual moistures from the
samelot of tissues,using their own techniques.
As seenin Table 5.2, measurementsby magnetic resonance
(MR) routinely gave higher values. Bank A, which used MR,
obtainedthe samevalue of 9.2Voforbothtypes of bone samples
becausethey usedonly one control. Measurementof eachtype
ofbone sampleby the gravimetric standardresultedin a value of
1.437o for cancellousbone and 6.98Vo for corticalbone,empha-
sizingthe needto include individual samplesof eachbone type.
Bank E usedthe Carl Fishermethod for residualmoisture. This
method is basedon a chemical reaction with extractablewater
resultingin a color change.It is accurateonly when the sample
can be solubilizedand thus is not a good procedurefor bone.As
can be seenin the table, the valueswere significantly lower by
this method than were valuesobtainedby the gravimetric stan-
dard.Bank B, which obtainedvery low valuesusingthe gravimet-
ric method, apparently did not dry their bone long enough to
reacha plateauofweight change.Even using a 90"C vacuum
oven and a desiccant,this processcan take over 200 hours to
achievea final equilibrium. Thus, standardizationof the gravi-
metric measurementmust be carried out.
Finally, to testfor maintenanceof packageintegrity, eachcon-
tainer shouldbe testedfor vacuum at the end ofthe freeze-drying
run as well as before shipment from the bank. This is usually
accomplishedusinga Teslacoil and looking for the generationof
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 FREEZE-DRYING 203

a color resulting from the excitation of gas molecules in the

container. This can only be done with bottled tissues;thus,
freeze-driedtissuesstored in plastic bags are usually given a
shorter expiration date, since the bags are likely to leak
over time.
Ultimately, the final quality control checkfor any freeze-dried
product should include a measurementof its function to deter-
mine that, in fact, preservationhastakenplace.In addition, such
measurementsshould be used to predict storagestability over
time. Quality control measurementsof this type are not rou-
tinely carried out by most tissuebanks,and an arbitrary expira-
tion dateof 5 yearshasbeenestablishedasthe standard(AATB).
It is very likely that there will be differencesin shelflife depend-
ing on storagetemperature and use (e.g., structural/osteocon-
ductive versusosteoinductivecapacityof freeze-driedbone).
Freeze-dryingis a complex processwith very practical appli-
cations. Performed properly, it servesas an ideal preservation
technique for long-term storageof various tissuesthat should
maintain their biologic and biomechanicalproperties.However,
this is possibleonly if appropriate precautions are taken and
good quality control is carried out.

ACKNOWLEDGMENTS

We thank the member organrzationsof the WesternAssocia-

tion of TissueBanks for providing information on their proce-
dures and tissuefor testing.Members who participated are the
Northern California Transplant Bank, the OregonTissueBank,
the Mile High Transplant Bank, the University of Texas,South-
westernMedical Center Transplant Services,and the American
Red Cross,St. Paul. We also thank Ms. Vicki Palmer and Ms.
and Ms. Kathy Ober-
Maria Austria for their editorial assistance
meyer and Ms. Margery Moogk for comments.

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204 FREEZE-DRYING

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