The Importance of Freezing on Lyophilization Cycle

Development

The freezing method

LThomas W. Patapoff and David E. Overcashier

used during lyophilization can

substantially affect the structure of the

ice formed, the water- vapor flow during

primary drying, and the quality of the final

dried product. Controlling how a

solution freezes can shorten lyophilization cycles and produce

more stable formulations.

16 BioPharm MARCH 2002

yophilization (freeze-drying) is oftenused to prepare dry pharmaceutical formulations to achieve commercially viable shelf lives. The process comprises three steps: freezing, primary drying, and secondary drying. As water freezes in the first step, the dissolved

components in the formulation remain in the resid- ual liquid, a phase termed the freeze-concentrate. At the point of maximal ice formation, the freeze- concentrate solidifies between the ice crystals that make up the lattice. Under appropriate lyophiliza- tion conditions, the ice is removed by sublimation during primary drying, leaving the remaining freeze-concentrate in the same physical and chemi- cal structure as when the ice was present. Residual water in the freeze-concentrate is removed in the secondary drying step.

Lyophilization cycle development typically focuses on optimizing the primary drying step. That is the most time consuming of the three steps, and the primary drying parameters are easily adjustable. They can affect both the time involved and the quality of the resulting cake. Extensive investigation of primary drying has demonstrated that two important parameters are chamber pressure and shelf temperature (1–9). They are usually adjusted to maximize the rate of heat transfer to each vial (speeding ice sublima- tion) without causing cake collapse.

Less attention has been paid to the freezing conditions and their potential effect on the pri- mary and secondary drying processes and on the characteristics of the final product. Kochs et al. reported the effects

of freezing conditions on pri- mary drying in a specially designed aluminum and plastic sample cell (10). They observed varia- tions in vapor diffusion coefficients (a measure of

the ease of water-vapor flow) as a function ofposition and cooling rate. The variations appeared to be largely due to variations in sample morphology. Searles et al. reported some effects of freezing on the rate of primary drying in vials (11). They found that the primary drying rate was dependent on the ice nucleation temperature. Faster drying was also attributed to an increase in lamellar ice crystal content, formed when ice nucleants from Pseudomonas syringae were added in the vial contents. Searles and colleagues also showed that adding a properly specified an- nealing step in the freezing procedure increased the drying rate (12).

Our article describes the effects of several freezing methods on water-vapor flow during pri- mary drying and on the final dried cake structure.

N O R M A L F R E E Z I N G A N D S U B L I M AT I O N When freeze-drying pharmaceutical products, manufacturers place vials containing aqueous formulations 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 that all the vials freeze completely. As the temperature decreases, the liquid cools below the freezing point of the solution: Supercooled temperatures range from 10 °C to 20 °C with ice nucleation typi- cally occurring at 15 °C. Freezing point depres- sion accounts for a lowering of the freezing tem- perature by only 1.8 kelvin

per molal. Most pharmaceutical manufacturing relies on supercool- ing because of the lack of nucleation centers in the formulations (such as particulates in solution or imperfections on the inside surface of the vials).

In a supercooled solution at 15 °C, ice can nucleate and propagate through the solution.

BioPharm MARCH 2002 17

Freezing Shelf Sublimation Product

Method 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

(a) Fast freezingStandard freezing

10,000 Vertical freezingAnnealed

8,000

6,000

4,000

2,000

0

(b)

50,000

Fast freezing40,000 Standard freezing

30,000Vertical freezingAnnealed

20,000

10,000

00 0.2 0.4 0.6 0.8 1.0

Dried Layer Thickness (cm)

then weighing the amount of the remaining solu- tion. We previously published a complete descrip- tion of this method for determining MTR (9).

Effect of the freezing method on MTR. Figure 1 shows MTRs during primary drying (10 °C shelf temperature, 100 mTorr chamber pressure) for vials frozen using the standard freezing process that we described earlier and for those frozen by alternative methods described below (plots 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 tubing vials). Following standard freezing, the resistance initially rose quickly, then more slowly as the sublimation front moved down the vial (increas- ing the thickness of the dried layer). That indi- cates that the upper layer of the dried cake contributed more to the resistance than did the lower portion, suggesting that the structure

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However, the entire solution cannot freeze imme- diately 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% crystal- lizes in multibranching, tortuous paths while the vial contents warm to approximately 1 °C. After the initial ice network has formed, addi- tional heat is removed from the solution by the shelf, and the remaining water freezes when the previously formed ice crystals grow.

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 chan- nels left by the sublimed ice above. Those narrow, often tortuous channels resist vapor flow, so subli- mation 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.

M A S S T R A N S F E R R E S I S TA N C E An important parameter governing the relation- ship between independent variables (the shelf temperature and chamber pressure) and depen- dent variables (the ice sublimation rate and the product temperature) has been defined by Pikal as the resistance to water-vapor flow or the mass transfer resistance (MTR) (2).

Determining MTR. MTR describes the propor- tionality between the specific sublimation rate m and the pressure driving force Po Pv:A p

Table 1. Effect of freezing method and primary drying shelf temperature on sublimation rate and primary drying product temperature

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

MTR =P o – P vm / A p

where m is the sublimation rate, Ap is the cross-sectional area of the product in the vial, MTR isthe normalized dried product resistance, Po is the equilibrium vapor pressure of ice at the tempera- ture of the subliming ice, and Pv is the pressure in the chamber. The relationship is analogous to that of electrical resistance: For a given pressure driving force (voltage), a decrease in MTR (elec- trical resistance)

BioPharm MARCH 200218

increases the rate of sublimation (electrical current). To determine MTR, measure the product temperature (from which we obtain Po using the equation published by Jancso et al., 13)and the sublimation rate m . These measurements can be obtained using a thermocouple and sequen- tially stoppering the vials during primary drying,

filled vial add fluorescent dye lyophilize

image under a

warm, add black paraffin under vacuum

cool, release

vacuum, the paraffin enters all the pores of the cake with no observable disturbance to the cake structure. The vial is then removed from the vacuum oven, cooled, and sectioned. The sectioned cakes are then observed under a fluorescence microscope. Interference from the fluorescence deeper in the cake is minimized by using a dark dye in the paraffin.

Correlating MTR to cake structure. The tortuous path that supercooled ice takes when propagating (Figure 3a) shows little directionality to the chan- nels after sublimation. Those channels, roughly25–100 m in diameter, serve as conduits for water-vapor transport from the sublimation front to the vial headspace. Resistance to vapor flowout of the cake is a result of both the small, tortu-

fluorescence microscope

cut plug

Figure 2. Paraffin dye method

plug ous channels and thick skin-like material on the top 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 the

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.

C A K E S T R U C T U R E The structure of a lyophilized cake has been reported several times (14–20). In most cases, cakes came from a supercooled solution with little or no attempt at controlling ice nucleation or propagation direction. Samples were generally cut or pulled apart and viewed by scanning electron microscopy (SEM). Because the potentially fragile cake structure was unsupported, the area exposed may have been altered by the cutting. If the cake is pulled apart, the tear will likely follow a structural weakness in the cake, so the exposed area may not be representative of the rest of the cake.

In the paraffin investment technique we devel- oped (Figure 2), paraffin acts as a support for the physical fine structure of the cake and

also prevents water absorption during sectioning, observation, and storage (9). The method uses a fluorescent molecule (such as rhodamine B) that is added to the solution before lyophilization and distributes equally throughout the cake (with no visible phase separation or spatial partitioning).

After lyophilization, the cake in the vial is placed under vacuum and warmed to 55 °C. Then paraf- fin is poured into the vial. While warm and under

sublimation front moves down through the first0.2 cm of the cake, corresponding to the thick- ness of the skin. Once the sublimation front passed the skin, the cumulative MTR increased more slowly (suggesting lower local MTR).

C O N T R O L L I N G I C E N U C L E AT I O N One way to produce directional freezing is to freeze the vial contents rapidly by immersing the vial into a dry-ice/ethanol bath until it is completely frozen. The resulting pores left from the rapid freezing and ice propagation are very small (Figure 3b). They start at the walls of the vial, move toward the middle, then move up. Although directional, the pores are small and nonvertical. The effect of such fast freezing can be seen in the MTR: The cumulative MTR increases rapidly and to a higher level (Figure 1a) and shows very high local MTR at small cake depths (Figure 1b). For fast freezing, that high MTR is attributable to the small size of the pores and to the horizontal direc- tion of the channels near the surface of the cake. The restricted vapor flow results in slower ice sub- limation.

Vertical freezing. To obtain straight, vertical channels, solutions were cooled on wet ice, nucleated at the bottom with dry-ice, and placed on a 50 °C shelf. The ice crystals propagate vertically, and no thick skin forms on the cake surface (Figure 3c). As expected, a vertical chan- nel orientation and the lack of thick skin on top lowers the MTR from that of samples from stan- dard and fast-freezing protocols (Figure 1a). Figure 1b shows that the local MTR is also low and constant for all cake depths. Interestingly, Figure 1b also suggests that changing the freezing

Table 2. Effect of freezingmethod on the dried state stability of a therapeutic proteina

Freezing AggregationMethod Rateb

Standard 2.3c

Annealed 1.6

a2.5 mg/mL protein, 123 mg/mLtotal excipient

bat 50 °Ccpercent per month

method did not alter the cake structure at the vial bottom, 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 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 dry- ing rates (11).

S U P E R C O O L I N G A N D A N N E A L I N G Ice structure and MTR during primary drying can be altered by freezing the vial contents using the standard method (supercooling followed by spon- taneous nucleation), then warming the frozen solution in the vial to a temperature just below the melting point (for example 2 ° C) for 10 hours. This allows the ice crystals to rearrange and grow to a more stable state. The vials are then cooled again to 50 °C. Figure 3d shows results from that annealing process. The cakes produced after annealing tend to have larger pores than those from the standard freezing method, which would be expected to facilitate water-vapor transmission and lower the MTR during primary drying. Figure 1a shows that the annealed material has a much lower MTR than solutions frozen by the standard or fast processes and is comparable to solutions that were nucle- ated 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.

D E C R E A S I N G M T R B Y O T H E R M E T H O D S Depending on solution composition, it may be possible to adjust the temperature (or the formu- lation) 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 small- scale collapse might alter the molecular structure or stability of the product would need to be eval- uated by an appropriate analytical program.

O P T I M I Z I NG T H E

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C Y C L E 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-

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.)

6,000

5,000

4,000

3,000

2,000

1,000

0

Active

Placebo

0 0.20.40.60.8

1Dried layer thickness (cm)

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 difficul

Figure 4. SEM micrographs of

t 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 (closerto 0 °C) by conditioning the bottom surface of the

freeze-

dried

materi

al and

the effe

ct of

formulation on

microstructure: (a) placeb

o (45

mg/mL

trehalose

,0.1 mg/mL polysorbate

20, and5 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 atGenentech, Inc., One DNA Way, South San Francisco, CA 94080,

650.225.8006, fax

650.225.7234,

tomp@gene.com,

www.gene.com.

Figure 5. Cumulative mass transfer resistance asa function of dried layer thickness for active and placebo material

perature for primary drying, which then matches that of a standard-frozen material dried at a colder shelf temperature. That higher shelf temperature adds driving force to the heat transfer, speeding primary drying. For equivalent product tempera- tures, the rate of sublimation was 40% faster for vertically oriented ice than

for standard-frozen so- lutions (Table 1). That leads to a decrease in the time required for primary drying — which consti- tutes half of the time spent in the lyophilization process. Such a decrease should also be expected from annealed vials, so the duration of the lyophilization cycle should be reduced as well.

O T H E R C O N S EQ U E N CE S O F F R E E Z IN G The freezing process can

have unexpected

conse- quences on

product quality. For

example, some forms

of sodium phosphate

can crystallize

upon slow freezing or annealing,

resulting in

a pH decrease in the freeze-

concentrate (21–23). A de-

crease in pH has been

shown to affect the stability of

a drug when frozen and

after lyophilization

(24–26). Other

excipients that crystallize

during freezing (for

example, mannitol) can

lose their abil- ity to stabilize proteins in the

dried state (27–30). Some

proteins undergo cold denaturation during slow

freezing or annealing,

which can have delete- rious

effects on product quality

upon reconstitution.

Freezing methods can

alter the void–solid

interfacial area of a lyophilized cake and, inversely, the thickness of its channel walls. An increased surface area correlates with decreased dried product stability for some proteins

(31). Freezing by immersing a vial into liquid nitrogen can result in increased protein aggregation (32) or decreased enzyme activity (33) when compared with freezer-cooled samples. Preliminary studies indicate that the freezing method has a significant effect on dried protein stability (Table 2). In addi-

glass vial. Nucleation would then occur on the bottom surface, and the ice would propagate in a vertical direction resulting in lower MTRs. Vials with that characteristic are not yet available. Annealing can be more practical but also has limitations. Whether annealing influences the

time for lyophilization or the temperature varia- tions of the product during freezing needs to be demonstrated.

Shortening the cycle.

The freezing method used during lyophilization can have a significant effect on the structure of the ice formed, affecting both the water-vapor flow during primary drying and the final product. Freezing protein solutions in

vials with vertically propagated crystals may prevent a thick skin from forming on the top of the lyophilized cake. The structure of vertically frozen material facilitates fast water-vapor flow during primary drying, resulting in lower product temperatures. By increasing the primary drying shelf temperature, the rate of sublimation was40% faster in our studies for the vertically frozen material than for

the standard-frozen material with an equivalent product temperature. We also observed increased sublimation in material annealed during the freezing segment. Decreased surface area correlates with increased product sta- bility for some proteins. Understanding and con- trolling how a solution freezes can lead to shorter lyophilization cycles and, in some cases, more stable products.

ACKNOWLEDGME

NTS The authors are indebted to Jamie Moore, MaryCromwell, Anne Nguyen, Al Stern, and Chung Hsu for technical assistance and consultation.

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BioPharm MARCH 200272

Validation

Importance of Freezing continued from page 21

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