Gap-Freezing Approach for Shortening the Lyophilization Cycle Time of Pharmaceutical Formulations—Demonstration of the Concept

Gap-Freezing Approach for Shortening the Lyophilization Cycle Time of Pharmaceutical Formulations—Demonstration of the Concept WEI Y. KUU,1 MARK J. DOTY,1 CHRISTINE L. REBBECK,1 WILLIAM S. HURST,1 YONG K. CHO2 1

Pharmaceutical Development, Baxter Healthcare Corporation, Round Lake, Illinois 60048

2

Bioscience Technical Services, Baxter Healthcare Corporation, Thousand Oaks, California 91362

Received 2 December 2012; revised 26 March 2013; accepted 25 April 2013 Published online 31 May 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.23610 ABSTRACT: During gap freezing, vials are placed on a metal tray, which is separated from the shelf surface with a small air gap that eliminates significant conductive heat transfer from the shelf to the bottom of the vial. The purpose of this freezing approach is to reduce the lyophilization cycle time of various amorphous formulations by nearly isothermal freezing. Such isothermal freezing promotes the formation of large ice crystals, and thus large pores throughout the cake, which subsequently accelerates the primary drying rate. The nucleation temperature using gap freezing, for the experimental conditions tested, was in the range of −1◦ C to −6◦ C, much higher than the range of −10◦ C to −14◦ C found using conventional shelf freezing. Isothermal freezing becomes effective when the gap is greater than 3 mm. The pore sizes and cake resistance during primary drying for various formulations were determined using the pore diffusion model developed by Kuu et al. (Pharm Dev Technol, 2011, 16(4): 343-357). Reductions in primary drying time were 42% (for 10% sucrose), 45% (for 10% trehalose), and 33% (for 5% sucrose). © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:2572–2588, 2013 Keywords: freeze-drying; lyophilization; diffusion; mathematical model; transport; algorithm; crystallization; porosity

INTRODUCTION Background of Nucleation and Crystallization Processes in Lyophilization Many aqueous pharmaceutical formulations are lyophilized to promote product stability. For these products, water is the first component to nucleate in the lyophilization cooling step, and then eventually freezes as the temperature is further lowered. The degree of supercooling of an aqueous solution is defined as the temperature difference between the thermodynamic freezing point of the solution and the temperature at which ice nucleation first occurs. The amount of supercooling is an important lyophilization parameter because it determines the number of ice nuclei formed and the ice crystal size in the frozen material.1–4 A lower degree of supercooling

Correspondence to: Wei Y. Kuu (Telephone: +224-270-5974; Fax: +224-270-5999; E-mail: wei [email protected]) Journal of Pharmaceutical Sciences, Vol. 102, 2572–2588 (2013) © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association

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(i.e., higher nucleation temperature) generates larger ice crystals, which eventually leave behind larger pores in the product cake after ice sublimation. These large pores are quite advantageous, in which cake mass transfer resistance is dramatically reduced, primary drying time is shortened, and low product temperature is maintained. Freezing is an important step in lyophilization because it impacts both process performance and product quality.5 The ice crystal structure, once formed, cannot be altered if not annealed, because the subsequent primary drying step only removes the ice via a combination of shelf temperature and chamber pressure. Thus, freezing should be performed with caution. Freezing can significantly impact the efficiency of primary and secondary drying phases. Hence, ultimately, it will have an impact on various product quality attributes such as morphology, physical state of the product, residual moisture content, and reconstitution time.5 It can also significantly impact the efficiency of the primary and secondary drying phases. Freezing is a challenging step in the lyophilization process because of its complex nature, and options

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for modifying conventional shelf-freezing approaches are often limited by the capability of the freeze dryer. A variety of techniques for inducing nucleation and freezing have been recently reviewed by Kasper and Friess,5 which include: (1) shelf-ramped freezing, (2) precooled shelf method, (3) ice fog technique, (4) electro-freezing, (5) ultrasound-controlled ice nucleation, (6) high-pressure shift freezing or depressurization technique, (7) quench freezing, (8) directional freezing, (9) addition of ice nucleating agents such as AgI, and (10) nonaqueous cosolvents, (11) annealing, and (12) vacuum-induced surface freezing, as discussed below. Approaches #1–#6 were developed to influence ice nucleation. In approach #6, the advantage of controlled nucleation, the termed ControLyoTM , is its ability to induce nucleation simultaneously in all vials on the entire shelf by pressurization and depressurization of the chamber, as described by Konstantinidis et al.6 In this article, it is reported that the technology is very effective for increasing the pore size in the cake for model formulations such as 5% mannitol, 5% sucrose, and a mixture of 3% and 2% sucrose. Note that the important upward freezing issue after nucleation, which is often serious to numerous formulations, was not addressed by the above nucleation approaches. When the formulations are difficult to freeze and even form a skin layer at the surface of the solution, nucleation is not the only issue during freezing. Also, in approach #4, electrofreezing, high voltage is used to induce ice nucleation. It has only applied in modified cryotubes, and it is not practical for manufacturing because each vial requires an electrode. In approach #7, quench freezing, vials are immersed into either liquid nitrogen or liquid propane. Although rapid freezing can quickly immobilize the solutes from migrating to the top of the solution, the ice crystals generated are generally small. Also, it is an uncontrollable freezing process, and very cumbersome in large-scale manufacturing. In approach #8, directional freezing, ice nucleation is induced at the bottom of the vial by contact with dry ice and followed by slow freezing on a precooled shelf. This approach would be difficult to implement in manufacturing. Approach #9 may not be acceptable for a pharmaceutical product because of the addition of the nucleation agent. Approach #10 is probably suitable for some formulations that already contains the nonaqueous cosolvents such as tertiary butyl alcohol, but not preferable for common aqueous pharmaceutical formulations because of the level of the residual solvent. In approach #11, annealing is a hold step at a temperature above the glass transition temperature (Tg  ). Annealing is a process step in which samples are maintained at a specified subfreezing temperature for a period of time to allow for complete crystallization of DOI 10.1002/jps

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crystalline compounds and to improve intervial heterogeneity and drying rates. Annealing has a rigorous effect on the ice crystal size distribution7,8 and can eliminate the interdependence between the ice nucleation temperature and ice crystal size and morphology. Annealing has dramatic effects on the particle size distribution of ice crystals for the following reasons. If the annealing temperature is above the Tg  , ice will melt as the system follows the freeze-point depression curve. Importantly, small ice crystals will melt faster than larger ones, and the small ice crystals may melt completely because of their higher free energy as compared with larger ice crystals.8–10 This Ostwald ripening (recrystallization) effect results in the growth of dispersed crystals larger than a critical size at the expense of smaller ones and is a consequence of these chemical potential driving forces.8,9 Small ice crystals do not reform upon refreezing of the annealed samples as the present large ice crystals serve as nucleation sites for continuous growth of ice crystals.8 Annealing can also result in the completion of freeze concentration (devitrification) by which exceeding the Tg  of an aqueous amorphous phase allows amorphous water to crystallize. Increased ice crystal size caused by annealing should accelerate primary drying. As a consequence, the intervial heterogeneity in ice crystal size distribution is reduced with increasing annealing time. Although annealing is commonly used in lyophilization recipes to allow for complete crystallization of crystalline solutes such as mannitol or glycine, we found that it cannot prevent the freeze concentration problem during the subsequent refreezing step because of upward freezing, especially in formulations that are difficult to freeze and/or with a large fill depth. Approach #12, vacuum-induced surface freezing, is not related to ice nucleation. It was described by Kramer et al.11 and later studied by Liu et al.12 It is able to prevent upward freezing and skin layer formation because freezing is induced at the surface of the solution. A concern of this method, as discussed by Kasper and Friess,5 is boiling of the formulation when it is still in the liquid state during vacuum induction. In the following sections, we will discuss how to use gap freezing to induce nearly isothermal freezing from the top and bottom of the solution to accelerate primary drying. Upward Freezing Phenomenon Using Conventional Shelf Freezing In conventional lyophilization, as depicted in the upper shelf of Figure 1, vials are placed directly on the shelf surface. Most laboratory-scale freeze dryers that are suitable for the development of lyophilization cycles for manufacturing have a shelf above every product shelf. The temperature of all shelves is JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 8, AUGUST 2013

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Figure 1. Schematic diagram of vials loaded on a gapped tray and a normal shelf in a chamber of a freeze dryer.

frequently controlled by a single circulating shelf fluid to maintain the shelf temperature. The typical serpentine flow patterns of the shelf fluid through the shelf channels is known from the literature.13 Because the bottom of the vial is in direct contact with the shelf surface, the heat transfer rate from the bottom shelf to the vial is normally much higher than that from the top shelf to the vial. This heat transfer differential causes nonuniform temperature distribution across the solution, especially during the cooling and freezing portion of the cycle when the shelf temperature is in the ramping mode. As a result, the solution at the bottom freezes much faster than that at the top. Moreover, as the solution on the bottom of the vial freezes, solutes are pushed upward in the vial, resulting in a solute concentration at the top of the vial, which can eventually form a “skin layer” at the cake surface.14 Conceivably, this issue could become more challenging as the fill volume increases.15,16 An example of this upward freezing phenomena using conventional shelf freezing is illustrated in Figure 2, where 7 mL of a 10% sucrose solution was filled into 20 mL glass tubing vials. These vials were placed close to the chamber door to easily photograph the freezing progress. As seen in Figure 2a, at time zero, prior to the nucleation, the solution is clear. After nucleation, the solution turns hazy, and with continuous cooling, forms a white layer of ice on the bottom of the vial as shown in Figure 2b. It is clear that the ice layer growth is in the upward direction. As freezing continues, the frozen layer of ice increases in height and pushes the unfrozen solutes to the top of the solution, as depicted in Figures 2c and 2d. Depending on the formulation composition, large amounts of these solutes, such as known amorphous components can conceivably be forced to the top of the solution. The freeze concentration effect shown in Figure 2a through Figure 2d will be similar at other locations on the shelf because heat conduction from the shelf JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 8, AUGUST 2013

surface to the bottom of the vial dominates the rate of heat transfer. This can also be verified by measuring the temperature profiles at the bottom and top of the solution in a vial (which will be discussed later in the text). Conceivably, the amorphous layer close to the top of the solution may become a substantial barrier to the sublimation of water vapor during primary drying. The photographs of vials during gap freezing are not shown in this report because they did not clearly show the expected “nearly equal freezing speed” of frozen ice layers at the top and bottom of the solution for the following reasons. To take good photographs of the freezing vials, it is necessary to place the vials very close to the front door. Therefore, these vials no longer have thermal shielding, which is necessary to represent a fully loaded shelf during lyophilization.17,18 Instead, the heat transfer process of these vials is similar to “single vials.” During shelf freezing, as shown in Figures 2a and 2b, this single vial effect has less impact on the progress of frozen ice layer because the heat transfer rate of conduction is much higher than convection and radiation.17,18 However, for vials during gap freezing, the impact of the front-door radiation could be more profound because of the absence of conduction. Therefore, additional radiation from the front door is significant enough to affect the freezing rate. As a result, the isothermal freezing effect of these single vials is weaker than that of the center vials because of the impact of the front-door radiation, which is confirmed by the temperature profiles measured at the top and bottom of the solution. In other words, the duration of isothermal freezing of the single vial during gap freezing is much shorter than that of fully loaded vials. Technical Rationale for Gap Freezing In gap freezing,19,20 as shown in the lower panel of Figure 1, the vials are resting on a metal tray with an air gap between the tray and the lower cooling shelf. The purpose of the gap is to control the heat conduction from the shelf surface to vials by separating the shelf and the tray a certain distance. The rate of heat transfer to the vial will be determined by the gap size and can be quite accurately controlled to obtain a desired rate. In fact, during the freezing step, the heat transfer rates to the top and bottom of the vial can be controlled to nearly equal, that is, nearly isothermal. During primary drying, the rate of heat transfer from the shelf to the vials will be much lower because the gap is an excellent insulator. However, it will be demonstrated later that the shelf temperature can be raised to a desired level to achieve the target production temperature. In other words, the cycle parameters of the shelf temperature and chamber pressure can be changed to optimize the cycle. This can be carried out using the approach proposed by Kuu and DOI 10.1002/jps

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Figure 2. Photographs of 10 mL of 10% sucrose in 20 mL tubing vials during freezing on a normal shelf: (a) at the onset and after 25 min after cooling, respectively, and (b) at 30 and 33 min after cooling, respectively.

Nail,21 if the overall heat transfer coefficient from the shelf, through the gap, to the vial can be determined. Therefore, the gap-freezing approach proposed here is capable of changing the conventional upward freezing to nearly isothermal freezing. Because of the unique heat transfer process, gap freezing can achieve either isothermal freezing at the top and bottom of the solution, or even moderate surface freezing if the gap size and shelf temperature are properly controlled. In addition, it also provides high nucleation temperatures that will be described below. Pore Diffusion Model for Determination of Product Resistance Parameters In this report, the effect of gap freezing on minimizing the product mass transfer resistance during primary drying will be determined using the pore diffuDOI 10.1002/jps

sion model proposed by Kuu et al.22 This model enables the determination of the resistance simply using the product temperature measured by thermocouples. The model assumes that diffusion of water vapor through the porous dry layer during primary drying follows the Knudsen regime of free molecular flow, where the Knudsen number is normally greater than 3.23 In this article, it was found that a simple modification to Knudsen diffusion coefficient using the pore radius equation, Eq. 1, rather than a constant pore radius, can fit the resistance well for a wide range of formulations and cycle conditions re /J = b0 + b1 

(1)

where re is the pore radius in cm; τ is the tortuosity factor, dimensionless. As the units on both sides of Eq. 1 need to be equal, the following units are JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 8, AUGUST 2013

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Table 1. Comparison of Resulting Mass Transfer Resistance Parameters for 10% Sucrose, 10% Trehalose, and 5% Sucrose Determined Using the Pore Diffusion Model, for Lyophilization Cycles Performed Using Shelf Freezing and Gap Freezing Cycle #

Formulation

b0 × 103 (cm)

b1 × 103 (Dimensionless)

1 (no gap) 2 (gap) 3 (no gap) 4 (gap)

10% sucrose 10% sucrose 5% sucrose 5% sucrose

0.131 0.238 0.101 1.83

0.797 1.44 2.30 2.76

Cycle # 5 (no gap) 6 (gap)

Formulation 10% trehalose 10% trehalose

b0 × 103 (cm) 0.154 0.303

b1 × 103 (Dimensionless) 0.310 0.786

RpN (m = 1.30 cm)a 8.1 4.4 2.81 1.45 RpN (m = 0.71 cm)aa 13.8 6.0

Tbmax b (◦ C) −33.5 −35.7 −32.6 −33.7 Tbmax b (◦ C) −31.6 −34.6

a  is the maximum cake thickness, which is estimated as: the fill volume divided by the internal cross-sectional area of the vial of 5.87 cm2 , followed by m the divide by 0.917 of the volume expansion factor because of freezing.18 bT bmax is determined by model extrapolation using the linear pore size equation, Eq. 1. Fill volume: 7 mL for 10% sucrose and 5% sucrose, and 3.8 mL for 10% trehalose. For 10% sucrose and 10% trehalose: primary drying performed using Tf = −25◦ C, Pc = 50 mTorr. For 5% sucrose: primary drying performed using Tf = −20◦ C, Pc = 100 mTorr, R0 = 0. Units of RpN : cm2 , torr, h, g−1 .

obtained: b0 is a constant, in centimeter, which has the same unit as re /τ; b1 is a constant, dimensionless, so that the combination (b1 ) has the same unit as re /τ;  is the dry layer thickness in centimeter. In addition, as per the reports by Pikal et al.,17,18 a nonzero intercept is also added to the resistance equation. Thus, the area-normalized resistance, RpN , is expressed by Eq. 2 22 [corrections added on 8th October 2013, after first online publication] 4.21 × 10−4 To g(b0 + b1 )

1/2

RpN = R0 +



(2)

where To is the average temperature in the dry layer, in K; ε is the porosity, dimensionless. In Eq. 2, the value of To is difficult to measure because of the moving boundary of the ice surface. However, the effect of To on RpN is not significant because of the fact that (RpN –R0 ) is proportional to the square root of To . For example, the value of To 1/2 only changes from 15.3 to 15.9 when To varies from −40◦ C to −20◦ C. As such, it can be assumed constant, for example, equal to 15.6. Note in Eq. 1 that when R0 = 0, re /τ becomes a quantitative expression for the pore radius by assuming a commonly used value for the tortuosity τ, such as 3.23 The detailed procedure for determination of the resistance parameters R0 , b0 , and b1 can be found in the literature.22 Using this model, the product temperature profile at the bottom–center, Tb (t), of the interior vial during primary drying alone can be used for determination of the pore size and mass transfer resistance.

MATERIALS AND METHODS Lyostar II Freeze Dryer, Vials, and Formulations Freezing studies and lyophilization cycle runs were performed using LyoStar II dryer (FTS Systems, SP Scientific, 3538 Main Street, Stone Ridge, New York, 12484, USA, www.SPScientific.com), in which the software Lyomanager II (FTS Systems) consists of JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 8, AUGUST 2013

16 drying steps. The freeze dryer has three shelves, with 16 thermocouple plugs for monitoring the product temperature. The chamber pressure was measured and recorded by a capacitance manometer, and the apparent pressures in the chamber and condenser were measured and recorded by Pirani gauges. Schott 20 mL tubing vials (Schott Pharmaceutical Packaging, Inc., Lebanon, Pennsylvania), cleaned using Miele professional G7883CD washer (http://mielepro.com/us/prof/home.html), were used for all freezing studies and lyophilization cycles. The internal and external cross-sectional areas of the vial are 5.87 and 6.95 cm2 , respectively, from which the fill depth of each freezing study and lyophilization cycle run can be quickly calculated. Various formulations were tested in this report, including the commonly used excipients for protein formulations: 10% (w/v) sucrose, 10% trehalose (w/v), and 5% (w/v) sucrose. Different fill volumes were used for various freezing studies and lyophilization cycles; however, the same volume was used in each group of comparison, as described below. For demonstration of upward freezing in Figures 2a and 2b, freezing studies for determination of nucleation temperature (Tn ) in Figure 6, and the freezing study of suspended vials in Figure 15, each vial was filled with 10 mL of the solution. The fill volume for 10% sucrose and 5% sucrose in Table 1 and the photographs in Figure 16 is 7 mL, whereas the fill volume for 10% trehalose in Table 1 is 3.8 mL. For determination of the lumped shelf-gap-tray heat transfer coefficient, KSGT , as depicted in Figures 13 and 14, each vial was filled with 7 mL of 5% mannitol. For demonstration of acceleration of drying rate by raising the shelf temperature using gap freezing, as presented in Figures 11 and 12, each vial was filled with 7 mL of 5% sucrose. All solutions were filtered with 0.2 :m filters. For each freezing study or lyophilization cycle run, the entire shelf was loaded with approximately 150 vials, and vials at the center location of the shelf were probed with 30 gauge Type T (copper/constantan) DOI 10.1002/jps

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Figure 3. Two thermocouples inserted in each probed vial to study the freezing process.

thermocouples.24 To perform freezing studies to evaluate the freeze concentration effect, two thermocouples were inserted in each probed vial, one at the bottom–center and the other approximately 2–3 mm below the liquid level, as shown in Figure 3. Thermocouples were calibrated to accuracy of ±0.5◦ C. Preparation of the Gap The gap can be easily prepared using low thermal conductivity spacers to separate the tray from the shelf surface. A caliper was used for accurate measurement of the gap because the size of the gap may have an impact on the heat transfer rate from the shelf surface to the tray. In Figure 1, the spacers are located at four

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Figure 5. Typical water–ice conversion process for vials on a 6 mm gapped tray during freezing. Solution: 10 mL of 10% sucrose. It can be seen that the temperature profile at the top of the solution (Ttop ) is lower and parallel to the profile at the bottom of the solution (Tbottom ).

corners underneath the tray on the lower shelf. For the purpose of stoppering of vials after lyophilization, spacers need to be uniformly distributed on the bottom surface of the tray. The tray was made of 1.5 mm thick 316 stainless steel sheet provided by the manufacturer of the LyoStar II freeze dryer (SP Scientific, 3538 Main Street, Stone Ridge, NY 12484, USA, www.SPScientific.com). Freezing Studies and Lyophilization Cycle Runs Using Gap Freezing and Shelf Freezing In the conventional lyophilization cycle, change between two temperature set points, Tf , is normally

Figure 4. Typical water–ice conversion process for vials during shelf freezing (without gap). Solution: 10 mL of 10% sucrose. It can be seen that the temperature at the bottom of the solution (Tbottom ) drops quickly after a short water–ice conversion period, which indicates faster freezing at the bottom. DOI 10.1002/jps

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Figure 6. Sumamry of Tn for various formulaiton using gap freezing and shelf freezing. The Tn value for each formulation includes the mean and standard deviation. Key: Su: sucrose, Tre: trehalose, NG: non-gap. The sample sizes (n) are: 10, 8, 7, 5, 6, 8, 7, and 6, in the order from left to right of the datasets in the figure.

Figure 7. Water–ice conversion index, θwic , as a function of the gap size, from 0 to 20 mm gap. Fill volume = 10 mL.

Figure 8. Comparison for the experimental and model-fitted product temperature profiles, Tb , for 10% trehalose, with gap freezing and shelf freezing (nongap). Fill volume = 3.8 mL. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 8, AUGUST 2013

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Figure 9. Comparison for effective pore radius for 10% trehalose, with gap freezing (6 mm gap) and shelf freezing (normal shelf). Fill volume = 3.8 mL; during primary drying, Tf = −25◦ C, Pc = 50 mTorr.

performed by a ramping step with a certain ramp rate in each step. Because of the absence of conduction in gap freezing, step change of Tf , instead of ramping, was used to shorten the freezing time because the insulation nature of the gap will prevent a rapid change in the product temperature. Step change of Tf means that the set point is “jumped” directly from one temperature to the other, and the actual changing rate of the product temperature, Tb (t), is determined by the cooling and heating capability of the freeze dryer. For example, with vials filled with 7 mL solution and resting on a 6-mm gapped tray, using a step change in Tf from 5◦ C to −70◦ C during freezing, the actual cooling rate in the vial is −0.55◦ C/min. By comparison for shelf freezing without a gap using the same step change in Tf , the actual cooling rate of Tb (t) with the same fill volume is −1.32◦ C/min. As expected, the actual cooling rate of Tb (t) using shelf freezing is much

higher than that of using gap freezing. Also note that the cooling rate of Tb (t) is a function of the gap size and fill volume. For the purpose of minimizing the number of cycles, if feasible, both the shelf freezing and gap freezing cycles were run simultaneously using the same freeze dryer and using the same step change in Tf . Because of the absence of thermal conduction from the shelf surface to the bottom of the glass vial, a low initial shelf temperature Tf , with the suggested range of −50◦ C to −70◦ C depending on the capability of the freeze dryer, is helpful for providing a sufficiently high cooling power to shorten the freezing time. For a lower fill depth, moderately low shelf temperature is sufficient. For a higher fill depth, a lower temperature is necessary. Likewise, during primary drying, the shelf temperature using gap lyophilization is generally much higher than that of

Figure 10. Comparison of resistance (RpN ) profiles for 10% trehalose using the cycles of shelf freezing (no gap) and gap freezing (gap). Fill volume = 3.8 mL; units of RpN : cm2 , torr, h, g−1 . DOI 10.1002/jps

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Figure 11. Effect of shelf temperature on product temperature profiles of the two 10% sucrose cycles: (1) gap freezing followed by shelf drying, and (2) gap freezing followed by gap drying. Fill volume: 7 mL filled in 20 mL tubing vials. Solid line: gap freezing followed by shelf drying, Tf = −25◦ C, Pc = 50 mTorr. Dotted line: gap freezing followed by gap drying, Tf = −5◦ C, Pc = 50 mTorr.

conventional lyophilization cycles. During secondary drying, because the thermal conductivity of the cake is low, the shelf temperature using gap lyophilization is similar to that used for shelf freezing and shelf drying. The following two experimental designs were used for the cycle runs. (1) For the purpose of determining the cake resistance in the gap-freezing cycle, the tray was

pulled out after the freezing step because the primary drying model22,25 was developed for the shelf-freezing cycle. The primary drying model for the gap-freezing cycle is yet to be developed. (2) However, for mimicking an actual gap-lyophilization cycle, spacers were left on the shelf to maintain a gap during primary drying and secondary drying.

Figure 12. Comparison of primary drying times for the two gap-freezing cycles in Figure 11, determined using Pirani gauge profiles.

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Figure 13. Product temperature profiles of 5% mannitol during primary drying using gap drying and shelf drying, following shelf freezing on both trays. Fill volume = 7 mL.

Freezing Study Using Suspended Vials to Mimic Gap Freezing Without Using a Tray To investigate whether the phenomena of high nucleation temperature and isothermal freezing during gap freezing were caused by the gap alone, a freezing study using suspended vials without a tray was also performed in this work. In this study, a metal frame for supporting the suspended vials was fabricated from test-tube racks. The diameter of the metal wires in the frame is approximately 1.8 mm, and dimensions of the openings of the frame are 3.5 × 3.5 cm2 . The thin frame was then placed on the shelf surface. To support the vials for maintaining a gap from the shelf surface, the frame was covered with an aluminum (Al) wire mesh screen. The approximate dimensions of the rectangular openings of the screen

are 1.5 × 2.0 mm2 . As the Al wire of the mesh is very thin, only 0.18 mm in diameter, most of the bottom surfaces of the vials are directly exposed to the shelf surface. The 20 mL glass vials were then placed on the screen and filed with 10 mL of 10% sucrose solution, with the approximately fill depth of 1.7 cm. Each of six center vials was probed with two thermocouples, one at the bottom–center and the other at approximately 2–3 mm below the liquid surface, as shown in Figure 3. The above vial and frame configurations should be suitable to simulate the heat transfer mechanisms of suspended vials. The distance (gap) from the shelf surface to the bottom of the vial is approximately 6 mm. The freezing procedure is the same as that used for Figure 5. Freezing was initiated by cooling the shelf from the ambient temperature to 5◦ C (by step change) and held at 5◦ C for 60 min, following

Figure 14. The experimental and model-fitted product temperature profiles, during gap freezing and gap drying, for determination of the lumped KSGT using 5% mannitol. Units of KSGT = cal, s−1 , cm−2 , ◦ C−1 . DOI 10.1002/jps

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Figure 15. Temperature profiles in suspended vials during freezing. Gap (from the shelf surface to the bottom of the vial) = 6 mm, fill volume = 10 mL. Key: TP (bottom): temperature at the bottom of the solution. TP (top): temperature at the top of the solution, approximately 2–3 mm below the liquid surface.

by cooling from 5◦ C to −70◦ C (by step change), and held at −70◦ C until the solution was solidified. Assessment of the Effectiveness of Gap Size on the Direction of Freezing The purpose of gap freezing is to minimize the upward freezing issue that hinders sublimation of water vapor. Because the freezing rate and the length of freezing time could vary among cycles, especially between shelf-freezing cycle and gap-freezing cycle, it is necessary to use a suitable indicator for comparison of the effect of gap size and freezing scheme on minimization of upward freezing. Thus, the water–ice conversion index, θwic , is defined here using the two temperature profiles at the top and bottom of the solution. The rationale of the index is that when the temperature profile is flat (constant), either at the top or bottom, it implies the coexistence of ice and water. On the contrary, when the temperature profile

drops quickly, it indicates that the conversion of water to ice is complete. The consequence of completion of water–ice conversion is that the growing ice layer will push unfrozen solutes away. If this event occurs at the bottom, the ice layer will grow upward, as illustrated by the photographs in Figures 2a and 2b. To elucidate the definition of the index, θwic , we are using the typical temperature profiles during cooling and freezing of 10% sucrose solution using shelf freezing in Figure 4, and using gap freezing in Figure 5. The preferable condition for freezing is that Ttop ≤ Tbottom , which implies that the unfrozen solutes would not be pushed upward to the top during freezing. For the case of shelf freezing in Figure 4, it is clear that Tbottom is lower than Ttop shortly after the completion of the water–ice conversion period. When Ttop starts to drop, it implies that the conversion at the top is complete. Thus, two tangent lines are drawn along the conversion phase and freezing phase of the curves. A vertical line is then drawn from the

Figure 16. Comparison of lyophilized cakes for 10% sucrose using gap freezing and shelf freezing. Fill volume = 7 mL; severe shrinkage in vials with shelf freezing is observed. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 8, AUGUST 2013

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intersection of these two lines. The integrated area, Area-2, is the area surrounded by the three lines, the conversion, the bottom, and the vertical lines. Area2 is treated as “negative” (unfavorable) because it is below the conversion line. Likewise, typical profiles of Tbottom and Ttop for gap freezing are presented in Figure 5. Using the same graphical procedure, the area surrounded by the three lines is Area-1, which is positive, because it is above Ttop . It should be noted that in many cases (not shown), especially during gap freezing, both Area-1 and Area-2 could exist in the temperature profiles during cooling and freezing. As per the above description, the “water–ice conversion index” is defined in Eq. 3. Water − ice conversion index = 2wic = ABC/(twic )top (3) where: twic , water–ice conversion time, in minute. (twic )top , water–ice conversion time at the top of the solution, in minute. (twic )bottom , water–ice conversion time at the bottom of the solution, in minute. ABC, area between curves, in ◦ C/minute, that is, the net area between the temperature profiles of the top (Ttop ) and bottom (Tbottom ) of the solutions during the water–ice conversion process. The value of ABC is positive when Ttop ≥ Tbottom , whereas it is negative when Ttop < Tbottom . Thus, the unit of θwic is ◦ C. The positive value of θwic is important for avoiding the possible freeze concentration and skin formation issues because of upward freezing. The ideal condition for avoiding these issues is 2wic ≥ 0 (4) For gap freezing, the value of θwic is a function of the gap size, which will be discussed later in the Results and Discussion section. Determination of Lumped KSGT The heat transfer coefficient of a normal shelf, Ks , of a LyoStar II freeze dryer (SP Scientific) has been determined by Kuu et al.13 using a temperature perturbation of the shelf fluid to create a nonsteady state heat transfer process. In this article, thermocouple junctions were placed on the surface of an empty and insulated shelf, where the insulation of the shelf was performed by evacuating the chamber to a low pressure, such as 100 mTorr, to minimize heat conduction by gas. In addition, the shelves were collapsed to minimize the sidewall radiation effect. The shelf surface temperature profiles along the serpentine flow path during the shelf temperature ramping period, Tsi (t), were then recorded. The shelf heat transfer coefficient was then determined from the two temperature proDOI 10.1002/jps

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files: the profile of Tsi (t) and the calculated shelf fluid temperature profile along the fluid path, Tf (t). For a laboratory freeze dryer, one of the simplest ways to minimize the sidewall radiation is by collapsing the shelves, as described above. Alternatively, we have tested that when the shelf surface is covered using insulation foam, the chamber could not reach a high vacuum level, such as below 300 mTorr. It was probably because of the continuous releasing of air from the pockets of the insulation foam. For our production freeze dryers (BOC Edwards freeze dryers; model Lyomax #9 and # 11, Crawley Business Quarter, Manor Royal, Crawley, West Sussex RH10 9LW, United Kingdom, http://www.edwardsvacuum.com), collapse of the shelves while running a cycle is not feasible because of the security lock of the system. However, in contrast to the laboratory freeze dryer, we also found that without collapsing the shelves, the entire shelf surface can be covered using a sheet of insulation foam to cut the sidewall radiation. After starting the vacuum pump, a high vacuum level as low as 30 mTorr can be easily reached. This is probably because of a different design of the vacuum pump of the production freeze dryer from that of the laboratory unit. The shelf temperature perturbation approach described above for determining the overall heat transfer coefficient using a gapped tray is not practical because the heat transfer processes using a gapped tray are more complicated. In this report, we used a simplified approach to quickly estimate the lumped shelf-gap-tray heat transfer coefficient, KSGT , which includes heat transfer from the shelf surface, to the tray, and then to the vial. In this experiment, two gapped trays, with a 6-mm gap beneath each tray, were loaded with 20 mL of Schott tubing vials, which were filled with 7 mL of 5% mannitol. The advantage of using mannitol is that the cake mass transfer resistance, RpN , is independent of the product temperature because of its crystalline nature.15,17,18 These gapped trays were placed onto two shelves of the LyoSrar II unit. Gap freezing for vials on both shelves was then performed by cooling the shelf from the ambient temperature to 5◦ C (by step change) and held for 60 min, followed by cooling from 5◦ C to −60◦ C (by step change), and held at −60◦ C until the solution was solidified. After complete solidification of the solution in the vials, the tray on the top shelf was removed to allow the vials in direct contact with the shelf surface to proceed shelf drying. Meanwhile, the other gapped tray was remained on the bottom shelf for gap drying. Primary drying was initiated by starting the vacuum pump with Tf = −15◦ C and Pc = 100 mTorr, followed by holding at −15◦ C, 100 mTorr, for 47 h. Secondary drying was conducted by holding at Tf = 30◦ C, Pc = 100 mTorr for 7 h. After completion of the cycle, the product temperature profiles, Tb (t), were JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 8, AUGUST 2013

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retrieved from LyoStar II system for determination of KSGT .

RESULTS AND DISCUSSION Typical Temperature Profiles During Freezing Typical temperature profiles, at the bottom and top of the solution, during shelf freezing and gap freezing are presented in Figures 4 and 5, respectively. In these figures, when the temperature stays constant over time, it implies coexistence of liquid water and frozen ice. It also suggests that the conversion process of water to ice is still ongoing. Conversely, when the water–ice conversion is complete, the temperature will start to drop. Figure 4 shows that for shelf freezing, after a short period of water–ice conversion, the temperature at the bottom starts to drop quickly, whereas the temperature at the top is still undergoing the conversion process for a much longer time. It is conceivable that a large amount of unfrozen solutes, especially the amorphous components, will be forced to the top during the conversion process. This is a typical freeze concentration effect because of upward freezing. By comparison in Figure 5 for gap freezing, both temperature profiles are parallel and stay flat for a long period of time. After completion of the conversion process, both temperatures drop nearly at the same time. It implies that the durations of water–ice conversion process at the bottom and top of the solution are similar. Hence, the freeze concentration phenomenon may be minimized because the unfrozen solutes will not be forced to the top to block the vapor flow during primary drying. Nucleation Temperature, Tn , of Various Cycle Runs For each lyophilization cycle run, the Tn was determined when the temperature started to spike during cooling, as shown in Figures 4 and 5. The resulting values of Tn for various cycle runs are presented in Figure 6, in which a common phenomenon is observed, that is, gap freezing results in significant increase of Tn , that is, significant reduction of the degree of supercooling. The range of Tn for gap freezing is from −1◦ C to −6◦ C, which is much higher than the range of −10◦ C to −14◦ C using shelf freezing. For the example of 10% sucrose, the Tn values using gap freezing and shelf freezing are −3.4 ± 1.5◦ C and −8.2 ± 2.0◦ C, respectively. It is surprising to observe that for some vials, the value of Tn is as high as −1◦ C. As discussed earlier that the degree of supercooling is important because it determines the number of ice nuclei formed at any time and thus the number and size of ice crystals in the frozen material. The degree of supercooling during the cooling and freezing steps of freeze-drying has a profound effect on the drying process and resulting cake structure, because JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 8, AUGUST 2013

it influences the ice structure. For example, a lower degree of supercooling (i.e., warmer nucleation) generates larger ice crystals, which leave behind larger pores as the ice is sublimed in primary drying. These larger pores can dramatically reduce the mass transfer resistance of the cake to shorten the drying time and lower the product temperature during primary drying. Although the exact cause of raising Tn is yet to be explored, it is probably because of the unique heat transfer process associated with gap freezing. Effect of Gap Size on Water–Ice Conversion Index The water–ice conversion index, θwic , between the bottom and top of the solution is very useful for assessing the extent of isothermal freezing. If θwic at the bottom solution is much smaller than that of the top solution, upward freezing is likely to occur. The typical temperature profiles at the bottom and top of the solutions, for 10% sucrose solution, are presented in Figures 4 and 5. These figures indicate that for vials resting on the shelf, the water–ice conversion time at the bottom of the solution is much shorter than that at the top. However, for vials resting on the 6-mm gapped tray, the water–ice conversion times for both thermocouple locations are very close. The effect of the gap size on θwic is shown in Figure 7, where for the vial resting on the 6-mm gapped tray, θwic is greater than 0. When the gap size decreases, the value of θwic appears to decrease. The value of θwic increases rapidly from shelf freezing, with gap thickness = 0–3 mm. When the gap size is greater than 3 mm, θwic approaches 0. The value of θwic appears to be the same as that at 6-mm gap when the gap is very large at 20 mm. It may be concluded from Figure 7, under the experimental conditions tested, that when the gap size is greater than 3 mm, the gap becomes effective for controlling the shelf heat transfer rate because the value of the index is close to zero. It should be noted that the most suitable application of the water–ice conversion index in Figure 6 is for comparison of the effect of the gap size on θwic for each container configuration, such as the vial size and fill volume. For a different configuration, it may be necessary to reconstruct Figure 7 experimentally, which can be carried out fairly quickly as compared with the lengthy lyophilization cycle. Various Lyophilization Cycle Runs Using Gap Freezing and Shelf Freezing Various lyophilization cycles were performed using gap freezing, including 10% sucrose, 10% trehalose, and 5% sucrose. For each cycle, the average product temperature versus time profile at the bottom– center of each probed vial, Tb (t), during primary drying was used to determine the mass transfer resistance and pore size using the pore diffusion model described earlier. Graphical illustrations of the DOI 10.1002/jps

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product temperature profile, pore radius, and mass transfer resistance RpN for 10% trehalose are presented in Figures 8–10, respectively. In Figure 8, the higher sublimation cooling effect of gap freezing, because of larger pores, can be seen from the much lower temperature profile, with the difference of approximately 2.2◦ C in the plateau region. As the gapped tray was pulled out after gap freezing for proceeding primary drying, the previously determined shelf heat transfer coefficient, Ks = 0.00378 cal s−1 cm−2 ◦ C−1 ,13 can also be used to determine the resistance parameters, R0 , b0 , and b1 , for vials using the gap-freezing cycle. Note in Eq. 2 that R0 is the intercept of the resistance (when the dry layer thickness is equal to 0), which is added to the resistance equation because of the deviation of the resistance to the Knudsen diffusion coefficient.17,18,22 For the cycles tested in Table 1, we found that the theoretical Tb (t) profile can closely fit the experimental values with R0 = 0. Thus, using the pore diffusion model for all cycles, the resulting resistance parameters b0 and b1 are presented in Table 1. The pore enlargement effect using gap-freezing for 10% sucrose and 10% trehalose in Table 1 can be easily seen from the much larger b1 values than those of shelf-freezing, since the other two parameters in Eq. 2, R0 and b0 , are nearly equal.22 The values of (1000 × b1 ) increase from 0.797 to 1.44 for 10% sucrose, and from 0.310 to 0.786 for 10% trehalose. For 5% sucrose in Table 1 using gap freezing, although the value of (1000 × b1 ) only slightly increases from 2.30 to 2.76, yet the value of (1000 × b0 ) dramatically increases from 0.101 to 1.83. The large b0 value of 1.83 for this particular gap freezing cycle suggests that the pores are large close to the surface of the cake. The pore enlargement effect because of gap freezing can also be assessed using the maximum product temperature, Tbmax , and the corresponding resistance, RpN . The model-extrapolated values of Tbmax of the gap-freezing cycles for 10% sucrose, 10% trehalose, and 5% sucrose are −35.7◦ C, −34.6◦ C, and −33.7◦ C, respectively. These values are much lower than the corresponding values of −33.5◦ C, −31.6◦ C, and −32.6◦ C, respectively, using the shelf-freezing cycle. The lower Tbmax is obviously because of larger pores that result in higher rate of sublimation cooling. The model-extrapolated RpN values for 10% sucrose, 10% trehalose, and 5% sucrose using gap-freezing cycles are 4.4 and 6.0, and 1.45 cm2 , torr, h, g−1 , respectively. These values are only about half of 8.1 and 13.8 and 2.81 cm2 , torr, h, g−1 of the corresponding shelf-freezing cycles, even with lower Tbmax values. For the purpose of graphical illustration, the profiles of pore radius and resistance, RpN , for 10% trehalose are presented in Figures 9 and 10, respectively. In Figure 10, the resistance is model-extrapolated using the linear pore size equation Eq. 1, to 0.71 cm of DOI 10.1002/jps

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the maximum cake thickness, as described above. As Figure 10 shows, using gap freezing, the maximum cake resistance is dramatically reduced from 13.8 to 6.0 cm2 , torr, h, g−1 . The estimated reductions in primary drying time, using the pore diffusion model, are 45% (for 10% trehalose), 42% (for 10% sucrose), and 33% (for 5% sucrose). These values suggest that the extent of reduction in primary drying time increases with the concentration of the amorphous solute, which is consistent with the rationale of gap freezing discussed earlier. Lumped KSGT The average product temperature profiles of the probed center vials on the two shelves, one with gap freezing followed by shelf drying, and the other with gap freezing followed by gap drying, are presented in Figure 13. As all vials were frozen using the same 6mm gapped trays, it is reasonable to assume that the pore sizes on both shelves are the same because of the same gap-freezing process. Also, as described earlier in the Materials and Methods section, the resistance of the mannitol cake is independent of the product temperature because of its crystalline nature. Thus, the dramatic difference in these profiles is attributed to the huge difference in the shelf heat transfer coefficient. As the heat transfer coefficient of the normal shelf, Ks , of the LyoStar II freeze dryer (SP Scientific) has been previously determined by Kuu et al.,13 the Tb (t) profile in vials using shelf drying can be used to determine the mass transfer resistance parameters R0 , b0 , and b1 , using the pore diffusion model.22 The resulting values are: R0 = 1.26 (cm2 , torr, h, g−1 ), b0 = 0.572 × 10−4 (cm2 , torr, h, g−1 ), and b1 = 0, where b1 = 0 confirms that the pore radius is independent of the dry layer thickness as per Eq. 1.18 Because the product temperature is a function of the dry layer thickness, it also suggests that the resistance profile is independent of the product temperature. Using these parameters, the nonlinear parameter estimate approach proposed by Kuu et al.25 was then used to search for the lumped shelf-tray heat transfer coefficient, KSGT . The experimental and model-fitted temperature profiles are depicted in Figure 14, in which the approximate value of KSGT is determined as 0.000201 cal s−1 cm−2 ◦ C−1 , which is smaller than 0.00378 cal s−1 cm−2 ◦ C−1 that was determined earlier by Kuu et al.13 using the shelf temperature perturbation approach. Figure 14 shows that the theoretical and experimental Tb (t) profiles fit closely nearly the entire range of primary drying prior to the breakout of Tb (t). Acceleration of Drying Rate for Gapped Tray by Raising the Shelf Temperature The lyophilization cycles using gap freezing discussed so far, as presented in Table 1 and Figures 8–10, are JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 8, AUGUST 2013

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gap freezing followed by shelf drying by pulling out the tray after completion of the freezing step. In this section, the purpose is to demonstrate the application of gap freezing to an actual lyophilization cycle, and to prove that the low heat transfer rate through the gap can be easily compensated by a higher shelf temperature. The two cycles demonstrated here are for 10% sucrose, with 7 mL filled into 20 mL tubing vials. The first cycle is gap freezing followed by shelf drying, with primary drying at the shelf inlet temperature Tf = −25◦ C and chamber pressure Pc = 50mTorr, and secondary drying at Tf = 30◦ C and Pc = 100 mTorr. The second cycle is gap freezing followed by gap drying, that is, without pulling out the tray after freezing, with primary drying at Tf = −5◦ C and Pc = 50 mTorr, and secondary drying at Tf = 35◦ C and Pc = 100 mTorr. The resulting product temperature profiles of the two cycles are depicted in Figure 11. The breakout of each product temperature profile suggests that the thermocouple junction was separated from the ice. The two groups of temperature profiles in Figure 11 indicate that when the shelf temperature is raised from −25◦ C to −5◦ C, the product temperature is much higher, which suggests a higher drying rate. In addition, the primary drying time for the cycle using Tf = −5◦ C appears to be shorter than that using Tf = −25◦ C simply from the much earlier breakout of the Tb profile. Comparison of the primary drying times for these two cycles, using the comparative pressure method of Pirani gauge versus the capacitance manometer, is shown in Figure 12. As seen, the primary drying time of the cycle with gap freezing and gap drying at Tf = −5◦ C is 41.5 h, which is much shorter than 62.5 h of the gap freezing and shelfdrying cycle at Tf = −25◦ C. The foregoing results indicate that the heat transfer rate from the bottom shelf to the vials on the gapped tray can be easily accelerated by raising the shelf temperature from −25◦ C to −5◦ C to achieve the desired product temperature. From the process modeling perspective, because the lumped KSGT has been determined, the cycle optimization approach proposed by Kuu and Nail21 can be used to quickly obtain the optimal combination of the shelf temperature and chamber pressure. Comparison of Gap Freezing of Vials with Freezing of Prefilled Syringes and Freezing of Suspended Vials Freezing studies in prefilled syringes have been reported by Hottot et al.26,27 and Patel and Pikal28 . In the second article by Hotott et al.,27 50 syringes were placed on a rack, whereas in the article by Patel and Pikal,28 two different syringe holders were used, that is, the plexiglass and Al block. In these three articles, syringes in the chamber of the freeze dryer are in the vertical tip-down position. As the bottom of the soluJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 8, AUGUST 2013

tion is not in direct contact with the shelf surface, a “gap” exists between the shelf surface and the solution. Hottot et al.27 reported that the nucleation temperatures during syringe freezing are similar to those during vial freezing. The authors observed that the lyophilized cakes in syringes are more uniform than those in vials. However, they explained that the phenomenon was probably because of the geometry of the syringe, that is, syringes are cooled simultaneously on three surfaces (top, bottom, and wall) and that the solution volumes in syringe configuration were smaller. In the third article by Patel and Pikal,28 the purpose of using Al block for syringe lyophilization studies is to accelerate the heat transfer rate. In this article, the degree of supercooling is highest in the vials followed by syringes in the Al block and then syringes in the plexiglass holder. The reasons for these differences are not completely clear but are likely related to the differences in cooling rates for the different product container configurations. Note in Figure 12 of the article28 that ice nucleation temperature from product thermocouple data ranges from −3◦ C to −12◦ C for the plexiglass holder and −10◦ C to −15◦ C for the Al Block. Also note in Figure 13 of the article28 that the distances from the shelf surface to the bottom of the syringe barrel of these two syringe holders are similar. Thus, the above description suggests that the difference in Tn in syringes using the two different holders may be because of the difference in heat transfer rate from the cylindrical side of the syringe, rather than the gap size. In this report, the purpose of the freezing study for suspended vials is to investigate the factors that influence the high Tn and isothermal freezing observed during gap freezing. The typical product temperature profiles at the top and bottom of the solution are presented in Figure 15. The values of Tn are in the range of −3.7◦ C to −7.7◦ C in the six probed vials, which seem to be similar to those during gap freezing in Figure 6. It should be noted that numerous freezing studies are necessary to determine a statistical significance of the comparison. Nevertheless, there is a common phenomenon for all probed vials after nucleation. As seen in Figure 15, after a short period of isothermal freezing, the temperature at the bottom of the solution falls quickly, whereas the temperature at top of the solution is still undergoing the water–ice conversion process. This is quite different from the temperature profiles depicted in Figure 5 using a 6-mm gapped tray to perform freezing. The above discussion suggests that to achieve isothermal freezing in vials, in addition to the gap, presence of the tray between the shelf and vials is critical. During gap freezing, the possible heat transfer processes include radiation, convection, and gas conduction through the gap.18 Presence of the tray will prevent a direct DOI 10.1002/jps

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radiative heat transfer from the shelf surface to the bottom of the vial. Appearance of Lyophilized Cakes Using Gap Freezing and Shelf Freezing Comparison of lyophilized 10% sucrose using both shelf freezing and gap freezing is shown in Figure 16, where shrinkage of cakes is clearly seen in vials lyophilized using the shelf-freezing cycle, which is probably in part because of microcollapse. By comparison, the cakes produced using gap freezing show absence of shrinkage. For some formulations, such as human serum albumin and sugars, the dramatic difference in cake structure between gap freezing and shelf freezing can be visually seen.

CONCLUSIONS The proposed approach successfully solves the conventional heat transfer problem during the freezing step of a lyophilization cycle that causes the ice layer growth in the upward direction. Using gap freezing, nearly isothermal freezing can be achieved, that is, the temperature profiles at the top and bottom of the solution are nearly equal. In addition, the nucleation temperature is much higher than that using the conventional shelf freezing, that is, much smaller degree of supercooling. As demonstrated by various formulations, gap freezing dramatically reduces the primary drying time, especially for highly amorphous formulations. The heat transfer rate from the bottom shelf to the vials on the gapped tray can be easily accelerated by raising the shelf temperature to achieve a desired product temperature. The metal tray appears to be a critical part of gap freezing because it serves as a heat transfer barrier to achieve nearly isothermal freezing. Because of the simplicity of this approach, the cost of implementing gap freezing is very low, especially for laboratory freeze dryers.

REFERENCES 1. Searles JA, Carpenter JF, Randolph TW. 2000. Primary drying rate heterogeneity during pharmaceutical lyophilization. Amer Pharm Rev 3:6–24. 2. Searles JA, Carpenter JF, Randolph TW. 2001. The ice nucleation temperature determines the primary drying rate of lyophilization for samples frozen on a temperature-controlled shelf. J Pharm Sci 90(7):860–871. 3. Rambhatla S, Ramot R, Bhugra C, Pikal MJ. 2004. Heat and mass transfer scale-up issues during freeze drying: II. Control and characterization of the degree of supercooling. AAPS Pharm Sci Tech 5(4):e58. 4. Pikal MJ, Rambhatla S, Ramot R. 2002. The impact of the freezing stage in lyophilization: Effects of the ice nucleation temperature on process design and product quality. Amer Pharm Rev 5:48–52. DOI 10.1002/jps

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5. Kasper JC, Friess W. 2011. The freezing step in lyophilization: Physico-chemical fundamentals, freezing methods and consequences on process performance and quality attributes of biopharmaceuticals. Eur J Pharm Biopharm 78:248– 263. 6. Konstantinidis AK, Kuu WY, Otten L, Nail SL, Sever RR. 2011. Controlled nucleation in freeze-drying: Effects on pore size in the dried product layer, mass transfer resistance, and primary drying rate. J Pharm Sci 100(8):3453–3470. 7. Schwegman, JJ, Carpenter JF, Nail SL. 2009. Evidence of partial unfolding of proteins at the ice/freeze-concentrate interface by infrared microscopy. J Pharm Sci 98 (9) 3239–3246. 8. Searles JA, Carpenter JF, Randolph TW. 2001. Annealing to optimize the primary drying rate, reduce freezing-induced drying rate heterogeneity, and determine Tg0 in pharmaceutical lyophilization. J Pharm Sci 90:872–887. 9. Searles JA. 2004. Freezing and annealing phenomena in lyophilization. In Freeze-drying/lyophilization of pharmaceutical and biological products; Rey L, May JC, Eds. New York: Marcel Dekker, Inc.. 10. Randolph TW, Searles JA. 2002. Freezing and annealing phenomena in lyophilization: Effects upon primary drying rate, morphology, and heterogeneity. Am Pharm Rev 5:40–47. 11. Kramer M, Sennhenn B, Lee G. 2002. Freeze-drying using vacuum-induced surface freezing. J Pharm Sci 91(2):433– 443. 12. Liu J, Viverette T, Virgin M, Anderson M, Dalal P. 2005. A study of the impact of freezing on the lyophilization of a concentrated formulation with a high fill depth. Pharm Dev Technol 10:261–272. 13. Kuu WY, Nail SL, Hardwick LM. 2007. Determination of shelf heat transfer coefficients along the shelf flow path of a freeze dryer using the shelf fluid temperature perturbation approach. Pharm Dev Technol 12:485–494. 14. Kasraian K, DeLuca P. 1995. The effect of tertiary butyl alcohol on the resistance of the dry product layer during primary drying. Pharm Res 12(4):491–495. 15. Pikal M.J, Shah S, Senior D, Lang J.E. 1983. Physical chemistry of freeze-drying: Measurement of sublimation rates for frozen aqueous solutions by a microbalance technique. J Pharm Sci 72(6):635–650. 16. Lu X, Pikal MJ. 2004. Freeze-drying of mannitol–trehalose— sodium chloride-based formulations: The impact of annealing on dry layer resistance to mass transfer and cake structure. Pharm Dev Technol 9(1):85–95. 17. Pikal MJ, Roy ML, Shah S. 1984. Mass and heat transfer in vial freeze-drying of pharmaceuticals: Role of the vial. J Pharm Sci 73:1224–1237. 18. Pikal MJ. 1985. Use of laboratory data in freeze drying process design: Heat and mass transfer coefficients and the computer simulation of freeze drying. J Parenter Sci Technol 9(3):115–138. 19. Kuu W. 2012. Optimization of nucleation and crystallization for lyophilization using gap freezing. Patent US2012/ 0077971A1, page 1 to 6 and sheet 1 to 9.. 20. Kuu WY, Doty MJ, Hurst WS, Rebbeck CL. 2012. Optimization of nucleation and crystallization for lyophilization using gap freezing. Patent US2012/0192448 A1, page 1 to 7 and sheet 1 to 14. Continuation-in-part of application no. 13/246,3432, filed on Sept. 27, 2011. 21. Kuu WY, Nail SL. 2009. Rapid freeze-drying cycle optimization using computer programs developed based on heat and mass transfer models and facilitated by tunable diode laser absorption spectroscopy (TDLAS). J Pharm Sci 98(9):3469– 3482. 22. Kuu WY, O’Bryan KR, Hardwick LM, Paul TW. 2011. Product mass transfer resistance directly determined during freezedrying using tunable diode laser absorption sepctroscopy JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 8, AUGUST 2013

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(TDLAS) and pore diffusion model. Pharm Dev Technol 16(4):343–357. 23. Ho NF, Roseman TJ. 1979. Lyophilization of pharmaceutical injections: Theoretical physical model. J Pharm Sci 68(9):1170–1174. 24. OMEGA Engineering, 1 Omega Drive, Stamford, Connecticut 06907-0047, http://www.omega.com. 25. Kuu WY, Hardwick LM, Akers MJ. 2006. Rapid determination of dry mass resistance for various pharmaceutical formulations during primary drying using product temperature profiles. Int J Pharm 313:99–113.

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26. Hottot A, Andrieu J, Vessot S, Shalaev E, Gatlin LA, Ricketts S. 2009. Experimental study and modeling of freeze-drying in syringe configuration. Part I: Freezing step. Dry Technol 27:40–48. 27. Hottot A, Andrieu J, Hoang V, Shalaev EY, Gatlin LA, Ricketts S. 2009. Experimental study and modeling of freeze-drying in syringe configuration. Part II: Mass and heat transfer parameters and sublimation end-points. Dry Technol 27:49–58. 28. Patel SM, Pikal MJ. 2010. Freeze-drying in novel container system: Characterization of heat and mass transfer in glass syringes. J Pharm Sci 99(7):3188–3204.

DOI 10.1002/jps