The Importance of Freezing on Lyophilization Cycle...
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the ease of water-vapor flow) as a function ofposition and cooling rate. The variationsappeared to be largely due to variations in samplemorphology. Searles et al. reported some effectsof freezing on the rate of primary drying in vials(11). They found that the primary drying rate wasdependent on the ice nucleation temperature.Faster drying was also attributed to an increase inlamellar ice crystal content, formed when icenucleants from Pseudomonas syringae wereadded in the vial contents. Searles and colleaguesalso showed that adding a properly specified an-nealing step in the freezing procedure increasedthe drying rate (12).
Our article describes the effects of severalfreezing methods on water-vapor flow during pri-mary drying and on the final dried cake structure.
NO R MA L FR EE Z ING A ND S U B L IMAT IONWhen freeze-drying pharmaceutical products,manufacturers place vials containing aqueousformulations on shelves within a lyophilizer. Typi-cally, the shelf temperature is then decreased (from5 °C) over three hours and held at a low tempera-ture (typically �40 °C to �50 °C) to ensure thatall the vials freeze completely. As the temperaturedecreases, the liquid cools below the freezing pointof the solution: Supercooled temperatures rangefrom �10 °C to �20 °C with ice nucleation typi-cally occurring at �15 °C. Freezing point depres-sion accounts for a lowering of the freezing tem-perature by only 1.8 kelvin per molal. Mostpharmaceutical manufacturing relies on supercool-ing because of the lack of nucleation centers in theformulations (such as particulates in solution orimperfections on the inside surface of the vials).
In a supercooled solution at �15 °C, ice cannucleate and propagate through the solution.
yophilization (freeze-drying) is oftenused to prepare dry pharmaceuticalformulations to achieve commerciallyviable shelf lives. The processcomprises three steps: freezing, primarydrying, and secondary drying. As waterfreezes in the first step, the dissolved
components in the formulation remain in the resid-ual liquid, a phase termed the freeze-concentrate.At the point of maximal ice formation, the freeze-concentrate solidifies between the ice crystals thatmake up the lattice. Under appropriate lyophiliza-tion conditions, the ice is removed by sublimationduring primary drying, leaving the remainingfreeze-concentrate in the same physical and chemi-cal structure as when the ice was present. Residualwater in the freeze-concentrate is removed in thesecondary drying step.
Lyophilization cycle development typicallyfocuses on optimizing the primary drying step.That is the most time consuming of the threesteps, and the primary drying parameters areeasily adjustable. They can affect both the timeinvolved and the quality of the resulting cake.Extensive investigation of primary drying hasdemonstrated that two important parameters arechamber pressure and shelf temperature (1–9).They are usually adjusted to maximize the rate ofheat transfer to each vial (speeding ice sublima-tion) without causing cake collapse.
Less attention has been paid to the freezingconditions and their potential effect on the pri-mary and secondary drying processes and on thecharacteristics of the final product. Kochs et al.reported the effects of freezing conditions on pri-mary drying in a specially designed aluminumand plastic sample cell (10). They observed varia-tions in vapor diffusion coefficients (a measure of
LThomas W. Patapoff and David E. Overcashier
The Importance of Freezing on Lyophilization Cycle
The freezing methodused during
lyophilization cansubstantially affectthe structure of the
ice formed, the water-vapor flow during
primary drying, andthe quality of the final
dried product.Controlling how a
solution freezes canshorten lyophilization
cycles and producemore stable
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BioPharm MARCH 2002 17
However, the entire solution cannot freeze imme-diately because the supercooled water can absorbonly 15 cal/g of the 79 cal/g of heat given off bythe ice formation. Therefore, the ice propagatesfrom the nucleation site, and about 19% crystal-lizes in multibranching, tortuous paths while thevial contents warm to approximately �1 °C.After the initial ice network has formed, addi-tional heat is removed from the solution by theshelf, and the remaining water freezes when thepreviously formed ice crystals grow.
Primary drying. At the completion of freezing,primary drying is initiated with the lowering ofthe chamber pressure and an increase in the shelftemperature. The ice sublimation front movesdownward through the frozen plug, a process thatmakes the water vapor transit through the chan-nels left by the sublimed ice above. Those narrow,often tortuous channels resist vapor flow, so subli-mation is slowed. Consideration of the vaporchannels illustrates how the method of freezing,by affecting ice crystal structure, can affectprimary drying. Decreasing the resistance to masstransfer of water vapor increases the sublimationrate, which leads to shorter primary drying timeswhile lowering the product temperature.
MA S S TRANSFER RES ISTANCEAn important parameter governing the relation-ship between independent variables (the shelftemperature and chamber pressure) and depen-dent variables (the ice sublimation rate and theproduct temperature) has been defined by Pikalas the resistance to water-vapor flow or the masstransfer resistance (MTR) (2).
Determining MTR. MTR describes the propor-tionality between the specific sublimation rate
and the pressure driving force Po�Pv:
where is the sublimation rate, Ap is the cross-sectional area of the product in the vial, MTR isthe normalized dried product resistance, Po is theequilibrium vapor pressure of ice at the tempera-ture of the subliming ice, and Pv is the pressure inthe chamber. The relationship is analogous to thatof electrical resistance: For a given pressuredriving force (voltage), a decrease in MTR (elec-trical resistance) increases the rate of sublimation(electrical current). To determine MTR, measurethe product temperature (from which we obtain Pousing the equation published by Jancso et al., 13)and the sublimation rate . These measurementscan be obtained using a thermocouple and sequen-tially stoppering the vials during primary drying,
MTR =P o – P vm / A p
then weighing the amount of the remaining solu-tion. We previously published a complete descrip-tion of this method for determining MTR (9).
Effect of the freezing method on MTR. Figure 1shows MTRs during primary drying (10 °C shelftemperature, 100 mTorr chamber pressure) forvials frozen using the standard freezing processthat we described earlier and for those frozen byalternative methods described below (plots used25 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 tubingvials). Following standard freezing, the resistanceinitially rose quickly, then more slowly as thesublimation front moved down the vial (increas-ing the thickness of the dried layer). That indi-cates that the upper layer of the dried cakecontributed more to the resistance than did thelower portion, suggesting that the structure of thedried layer varied in the vertical direction.
Curves in Figure 1a represent the effect of thedried layer thickness on the cumulative MTR. The
Figure 1. Mass transferresistance as a function of driedlayer thickness for materialfrozen by various methods: (a)cumulative MTR, (b) derivativeof MTR
0 0.2 0.4 0.6 0.8 1.0
Dried Layer Thickness (cm)
Fast freezingStandard freezingVertical freezingAnnealed
Fast freezingStandard freezingVertical freezingAnnealed
Table 1. Effect of freezingmethod and primary drying shelftemperature on sublimation rateand primary drying producttemperature
Freezing Shelf Sublimation ProductMethod Temp (°C) Rate (g/hr) Temp (°C)
Standard 10 0.36 �24.5
Vertical 10 0.46 �27.0
Vertical 20 0.48 �26.0
Vertical 30 0.55 �23.5
vacuum, the paraffin enters all the pores of thecake with no observable disturbance to the cakestructure. The vial is then removed from thevacuum oven, cooled, and sectioned. Thesectioned cakes are then observed under afluorescence microscope. Interference from thefluorescence deeper in the cake is minimized byusing a dark dye in the paraffin.
Correlating MTR to cake structure. The tortuouspath that supercooled ice takes when propagating(Figure 3a) shows little directionality to the chan-nels after sublimation. Those channels, roughly25–100 �m in diameter, serve as conduits forwater-vapor transport from the sublimation frontto the vial headspace. Resistance to vapor flowout of the cake is a result of both the small, tortu-ous channels and thick skin-like material on thetop of the cake (reaching a thickness greater than1–2 mm). In the standard freezing scenario ofFigures 1a and 1b, high MTR can be seen as thesublimation front moves down through the first0.2 cm of the cake, corresponding to the thick-ness of the skin. Once the sublimation frontpassed the skin, the cumulative MTR increasedmore slowly (suggesting lower local MTR).
C O NTR O LL ING I C E NU C LEAT IO NOne way to produce directional freezing is tofreeze the vial contents rapidly by immersing thevial into a dry-ice/ethanol bath until it is completelyfrozen. The resulting pores left from the rapidfreezing and ice propagation are very small (Figure 3b). They start at the walls of the vial,move toward the middle, then move up. Althoughdirectional, the pores are small and nonvertical.The effect of such fast freezing can be seen in theMTR: The cumulative MTR increases rapidly andto a higher level (Figure 1a) and shows very highlocal MTR at small cake depths (Figure 1b). Forfast freezing, that high MTR is attributable to thesmall size of the pores and to the horizontal direc-tion of the channels near the surface of the cake.The restricted vapor flow results in slower ice sub-limation.
Vertical freezing. To obtain straight, verticalchannels, solutions were cooled on wet ice,nucleated at the bottom with dry-ice, and placedon a �50 °C shelf. The ice crystals propagatevertically, and no thick skin forms on the cakesurface (Figure 3c). As expected, a vertical chan-nel orientation and the lack of thick skin on toplowers the MTR from that of samples from stan-dard and fast-freezing protocols (Figure 1a).Figure 1b shows that the local MTR is also lowand constant for all cake depths. Interestingly,Figure 1b also suggests that changing the freezing
local MTR for various cake depths can be determined from the derivative of those curves(Figure 1b). The derivative curve (for standardfreezing) also shows that the upper layer of thedried cake contributes more to the resistance thandoes the lower portion. That result is consistentwith the results published by Kochs et al. (10).The relationship between resistance and driedcake structure can be investigated by studyingcake structure after lyophilization.
CAKE STRUCTUREThe structure of a lyophilized cake has beenreported several times (14–20). In most cases,cakes came from a supercooled solution with littleor no attempt at controlling ice nucleation orpropagation direction. Samples were generally cutor pulled apart and viewed by scanning electronmicroscopy (SEM). Because the potentially fragilecake structure was unsupported, the area exposedmay have been altered by the cutting. If the cake ispulled apart, the tear will likely follow a structuralweakness in the cake, so the exposed area may notbe representative of the rest of the cake.
In the paraffin investment technique we devel-oped (Figure 2), paraffin acts as a support for thephysical fine structure of the cake and alsoprevents water absorption during sectioning,observation, and storage (9). The method uses afluorescent molecule (such as rhodamine B) thatis added to the solution before lyophilization anddistributes equally throughout the cake (with novisible phase separation or spatial partitioning).After lyophilization, the cake in the vial is placedunder vacuum and warmed to 55 °C. Then paraf-fin is poured into the vial. While warm and under
Figure 2. Paraffin dye method
warm, add black paraffin under vacuum
filled vial add fluorescent dye lyophilize
image under afluorescence microscope
cool, release plug
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method did not alter the cake structure at the vialbottom, because the local MTR values are equiva-lent for dried layer thickness greater than 0.5 cm.
Vertical freezing produces a higher sublima-tion rate and lower product temperature duringprimary drying (Table 1). The lower MTR allowsfaster sublimation, which results in lower producttemperatures through increased heat removalfrom the latent heat of sublimation. The verticalfreezing results are consistent with those foundby Searles et al. that the presence of lamellar (instead of spheroidal) ice crystals increases dry-ing rates (11).
S U P E R COOL ING AND ANNEAL INGIce structure and MTR during primary drying canbe altered by freezing the vial contents using thestandard method (supercooling followed by spon-taneous nucleation), then warming the frozensolution in the vial to a temperature just belowthe melting point (for example �2 °C) for 10 hours. This allows the ice crystals to rearrangeand grow to a more stable state. The vials arethen cooled again to �50 °C. Figure 3d showsresults from that annealing process. The cakesproduced after annealing tend to have largerpores than those from the standard freezingmethod, which would be expected to facilitatewater-vapor transmission and lower the MTRduring primary drying. Figure 1a shows that theannealed material has a much lower MTR thansolutions frozen by the standard or fast processesand is comparable to solutions that were nucle-ated on the bottom with vertical ice propagation.The lower resistance observed for annealedmaterial is consistent with a faster sublimationreported by Searles et al. (12). Figure 1b alsoshows that the annealed material has a local MTRthat is low and constant throughout the cake. Theeffects of any skin appear to be minimal.
D E C R E A S ING MTR BY OTHER METHODSDepending on solution composition, it may bepossible to adjust the temperature (or the formu-lation) such that primary drying takes place atproduct temperatures near the glass transitiontemperature (Tg�) of the freeze-concentrate. Thatallows the freeze-concentrate to flow enough tocreate holes in the channel walls so water vaporcan be transmitted in a less tortuous path, moredirectly up and out of the cake.
Figure 4a shows an SEM result in which holeshave formed in the walls of the pores. Theseporous walls were not evident in formulationscontaining protein (Figure 4b) but were observed
only in formulations using a protein-free placebo,which has a lower Tg�. The holes were lessevident in the protein-free material when it wasdried at lower temperatures. That implies that notall formulations are amenable to pore formation.
Figure 5 shows that the MTR for the placebowas significantly lower than that of the solutionthat contains protein (9). Obviously, the selectionof processing conditions needs to consider finalproduct quality. The possibility that such a small-scale collapse might alter the molecular structureor stability of the product would need to be eval-uated by an appropriate analytical program.
OPT IM I Z ING TH E C Y C LEThe manner of freezing can have a significantimpact on primary drying characteristics of aformulation. For a given primary drying shelftemperature and chamber pressure, a decrease inMTR will increase the rate of sublimation. Inaddition, an increase in the rate of sublimationwill lead to a lower product temperature becauseof the enthalpy of ice sublimation (Table 1).Increasing the primary drying shelf temperature ofvertically frozen vials increases the product tem-
Figure 3. Freeze-dried cakeappearance corresponding tostandard and alternativefreezing methods: (a) supercooling using standardfreezing; (b) fast freezing; (c) vertical freezing; and (d) standard freezing followedby annealing (photo courtesy ofGenentech, Inc.)
Table 2. Effect of freezingmethod on the dried statestability of a therapeutic proteina
Freezing AggregationMethod Rateb
a2.5 mg/mL protein, 123 mg/mLtotal excipient
bat 50 °Ccpercent per month
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Figure 4. SEM micrographs offreeze-dried material and the
effect of formulation onmicrostructure: (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/mLtrehalose, 0.1 mg/mL
polysorbate 20, and 5 mMhistidine at 6.0 pH) (photo
courtesy of Genentech, Inc.)
perature for primary drying, which then matchesthat of a standard-frozen material dried at a coldershelf temperature. That higher shelf temperatureadds driving force to the heat transfer, speedingprimary drying. For equivalent product tempera-tures, the rate of sublimation was 40% faster forvertically oriented ice than for standard-frozen so-lutions (Table 1). That leads to a decrease in thetime required for primary drying — which consti-tutes half of the time spent in the lyophilizationprocess. Such a decrease should also be expectedfrom annealed vials, so the duration of thelyophilization cycle should be reduced as well.
OTHER CONSEQUENCES O F FR EE Z INGThe freezing process can have unexpected conse-quences on product quality. For example, someforms of sodium phosphate can crystallize uponslow freezing or annealing, resulting in a pHdecrease in the freeze-concentrate (21–23). A de-crease in pH has been shown to affect the stabilityof a drug when frozen and after lyophilization(24–26). Other excipients that crystallize duringfreezing (for example, mannitol) can lose their abil-ity to stabilize proteins in the dried state (27–30).Some proteins undergo cold denaturation duringslow freezing or annealing, which can have delete-rious effects on product quality upon reconstitution.
Freezing methods can alter the void–solidinterfacial area of a lyophilized cake and,inversely, the thickness of its channel walls. Anincreased surface area correlates with decreaseddried product stability for some proteins (31).Freezing by immersing a vial into liquid nitrogencan result in increased protein aggregation (32) ordecreased enzyme activity (33) when comparedwith freezer-cooled samples. Preliminary studiesindicate that the freezing method has a significanteffect on dried protein stability (Table 2). In addi-
tion, reconstitution time could be affectedbecause increasing the surface area may allowfaster reconstitution.
P R A C T IC A L A S P EC TSFreezing methods are seldom investigatedbecause freezing is difficult to control. Normally,pharmaceutical products supercool to roughly�15 °C before nucleating, which is difficult toovercome by standard processes. Ice might beinduced to nucleate at higher temperatures (closerto 0 °C) by conditioning the bottom surface of theglass vial. Nucleation would then occur on thebottom surface, and the ice would propagate in avertical direction resulting in lower MTRs. Vialswith that characteristic are not yet available.Annealing can be more practical but also haslimitations. Whether annealing influences thetime for lyophilization or the temperature varia-tions of the product during freezing needs to bedemonstrated.
Shortening the cycle. The freezing method usedduring lyophilization can have a significant effecton the structure of the ice formed, affecting boththe water-vapor flow during primary drying andthe final product. Freezing protein solutions invials with vertically propagated crystals mayprevent a thick skin from forming on the top ofthe lyophilized cake. The structure of verticallyfrozen material facilitates fast water-vapor flowduring primary drying, resulting in lower producttemperatures. By increasing the primary dryingshelf temperature, the rate of sublimation was40% faster in our studies for the vertically frozenmaterial than for the standard-frozen materialwith an equivalent product temperature. We alsoobserved increased sublimation in materialannealed during the freezing segment. Decreasedsurface area correlates with increased product sta-bility for some proteins. Understanding and con-trolling how a solution freezes can lead to shorterlyophilization cycles and, in some cases, morestable products.
A C K NO WLED G MENTSThe authors are indebted to Jamie Moore, MaryCromwell, Anne Nguyen, Al Stern, and Chung Hsu fortechnical assistance and consultation.
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Figure 5. Cumulative mass transfer resistance asa function of dried layer thickness for active andplacebo material
0 0.2 0.4 0.6 0.8 1Dried layer thickness (cm)
Corresponding author Thomas W.Patapoff is a senior scientist and
director, and David E. Overcashieris 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.
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Importance of Freezing continued from page 21
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