Stefan Schneid works in the Department of Pharmaceutics at the University of Erlangen in Erlangen, Germany.

Stefan Schneid

The pharmaceutical industry and the US Food and Drug Administration have come to realize that testing quality into a final product can inhibit the rate of introducing new drugs to the market. Since 2003, FDA has been working to modernize pharmaceutical manufacturing and encouraging the use of process analytical technology (PAT) and quality by design (QbD) under its Pharmaceutical CGMPs for the 21st Century initiative (1). The goal of the initiative is to enable manufacturers to produce drug products more efficiently and with higher quality. To achieve this goal, manufacturers must specify critical process parameters (CPPs) and control them within predefined limits of the so-called "design space" (2). These specifications, however, must be developed with laboratory experiments using PAT tools that have the capability to measure CPPs with acceptable accuracy (3). With freeze-drying, in particular, the use of PAT in the laboratory is more focused on process understanding, whereas PAT in production-scale freeze-drying is necessary for process control and may expedite scale-up. The "SMART" freeze-dryer concept (FTS Systems, Stone Ridge, NY) which is based on manometric temperature measurement (MTM), is a new PAT tool that allows accurate product temperature determination at the ice sublimation interface (

) and the collection of product resistance data (

) during primary drying (4–6). In addition, SMART technology provides an optimized cycle recipe during the first laboratory experiment for a given formulation. This article describes an approach to evaluate the robustness of a product based on a few simple laboratory experiments for the purposes of scaling-up and to study the impact of a potential 

Based on an optimized cycle recipe for 50 mg/mL sucrose, shelf temperature and pressure settings were varied to simulate potential worst-case conditions during a production scale run. 

 data were collected during the laboratory experiments using an auto-MTM procedure and correlated with  the product's appearance. The sucrose purchased from Sigma-Aldrich (Munich) was of analytical grade and used as received. Solutions of 50, 100, and 200 mg/mL were prepared with double-distilled water from an all-glass apparatus.  

Freeze-drying was performed with a "FTS Lyostar II" laboratory-scale freeze dryer with the latest version of SMART freeze-dryer software, which also collects MTM data for a user-predefined recipe (Auto-MTM). Three milliliters of solution were filled into 112 20-mL serum tubing vials (Wheaton, Millville, NY) (vial inner area: 5.74 cm

). Vials were placed hexagonally on the middle shelf of the freeze dryer, using one row of empty vials for shielding. Aluminum foil was placed adjacent to the front door inside the chamber to minimize radiation effects. The first cycle used SMART software to obtain a cycle recipe for the product and experimental conditions (

= –32 °C, safety margin: 2 °C). The cycle recipe obtained from the SMART run was adjusted to facilitate scale-up conditions (constant shelf temperature over time profile during primary drying). Subsequent runs were performed based on this improvement and then modified for shelf temperature (

: 62–200 mTorr), and solute concentration (50–200 mg/mL). 

 data were recorded and analyzed to describe the influence of the process variations and total solid content on the product morphology.   

) were determined using a "FDCS 196" freeze-drying stage (Linkam Scientific Instruments, Surrey, UK) and an "Axio Imager" microscope (Zeiss, Göttingen, Germany). Images were captured with a 1.3 MPix digital camera and analyzed using "LinkSys" (Linkam) software. The protocol applied was a standard procedure reported in the literature (7, 8). Onset of collapse for the sucrose was determined as follows:  

 Lyophilized samples were broken into pieces, fixed on aluminum stubs and then carefully gold-sputtered at 20 mA/5 kV (Hummer JR Technics) for about 30 s. Cake morphology was then examined using an Amray 1810 T scanning electron microscope (SEMTech Solutions, CITY, MA) at 20 kV. 

Cycle design by manometric temperature measurement.

 Figure 1 illustrates the cycle recipe obtained for 50 mg/mL sucrose. The recipe is optimized for the type of excipient, individual 

 of the product, and container system. Note that 

 was automatically lowered by the software after about 6 h to account for a continuous increase in  

 toward the end of primary drying, caused by an increase in 

 (see Figure 4). Product temperature at the sublimation interface did not exceed the collapse temperature during primary drying (see Figure 3 and Table I). A freeze-drying cycle may be denoted as "optimized" when  

 (i.e., 2–3 °C) during primary drying to avoid structural loss (9). For the SMART cycle, total primary drying time during this run was 1136 min and the cycle recipe in primary drying as follows: –39 °C (60 min), –18 °C (358 min), –23 °C (718 min). The endpoint of primary drying was determined using MTM and based on the total difference between the calculated P

value by MTM (nonlinear regression analysis of the pressure rise data) and the chamber pressure, 

. A representative determinant of the endpoint of primary drying is crucial for testing robustness as described in this article. Once all ice has  been removed, the pressure rise in the chamber during an MTM measurement is based on leaks and additional heat transfer to the product. Thus, the "fitted" vapor pressure of ice obtained from the MTM procedure equals that of the chamber pressure (see Figure 1) (3–5).  

 over time (averaging of the SMART shelf-temperature settings during primary drying to obtain a constant shelf temperature over time) and 

 from 52 to 62 mTorr) was conducted to further improve the recipe and to facilitate scale-up (see Figure 2) (10).  

 profiles over time for this modified cycle were found in very good agreement to the initial SMART cycle with a maximum temperature of –34.8 °C during the run (see Figure 3 and Table I). Total primary drying time was 1190 min, or about 5% longer than the initial cycle recipe. It is important to note that the product processed in both cycles showed no indication of shrinkage or collapse (see Figure 5a).  

 For 50 mg/mL sucrose, variation of shelf temperature of ± 5 °C had very little influence on the profile of  

 over time (see Figure 3). However, an increase in 

 from 52 to 200 mTorr led to a significant increase in  

. In contrast, an increase in solid content to 200 mg/mL and use of identical process conditions revealed only an increase of 1 °C in  

Theoretical modeling of the freeze-drying cycle is an alternative method to gain a better understanding of the impact of process variables on the product-temperature profile. A one-dimensional steady state of the freeze-drying process was used to simulate the influence of 

 for the 50 mg/mL sucrose solution. Note that 

 parameters were used according to the experimental design described above (see Figure 3 and Table I). During primary drying, the system is in a "pseudo steady state" and the following equation can be applied to predict product temperature (3, 11): 

 is the heat of sublimation of ice (670 cal/g used as constant), 

 is the vapor pressure of ice (Torr, known from the first experiment), 

 are the product and stopper resistance, respectively (cm

 ×Torr ×h/g, known from the first experiment), 

 is the vial outer cross-sectional area (6.83 cm

 is the vial heat transfer coefficient (10

 is the shelf-inlet temperature (°C) and T

 the temperature at the bottom of the vial (°C).  

Table I illustrates the theoretical values obtained from the steady-state model for the maximum product temperature (

) when using a ± 5 °C shelf temperature variation at constant pressure (62 mTorr) or an increase in chamber pressure from 62 to 200 mTorr at constant 

Table I: Summary of process conditions for 50â200 mg/mL sucrose solutions and corresponding maximum product temperature data (Tp-max) during primary drying.  

 were found in fairly good agreement to the experimental data. Note that this observation has been reported in the literature (11). However, the effect of 

 on product temperature was lower in the experiments performed relative to the theoretical calculations, in particular for applied pressure changes. Although such simulation is useful for scale-up, rational design of the freeze-drying process and troubleshooting, simulations cannot eliminate the need for careful experimentation (12). 

 data determined for products dried by the optimized cycle increased almost linearly over L

, indicating an absence of shrinkage and microcollapse (see Figure 4) (4, 5). The variation of 

. However, the pressure increase up to 200 mTorr resulted in a significant decrease in 

. Microcollapse as well as shrinkage in these products could be detected visually and by SEM (see Figure 5b).  

Note that an increase in solute concentration led to a similar 

 curve shape relative to the samples at high 

, but the product matrix revealed much greater robustness to elevated temperature (see Figures 4 and 5c). This observation was in excellent agreement with the 

 difference measured by FDM between 50 mg/mL (

 = –32 °C) sucrose solutions. Note that the dependency of sucrose concentration on 

 was recently investigated but is in contrast to data obtained in early studies on a different amorphous system (8, 13). 

The study indicates that safety margins for shelf temperature and chamber pressure for a given product can be pre-evaluated in a laboratory using advanced PAT tools and therefore may allow for a rational QbD approach in the future. In addition, product resistance data obtained from MTM may provide valuable information about microcollapse in the product ("benchmark" properties). This information can be very useful in interpreting the effect of temperature or pressure deviations on a product. 

 work in the Division of Pharmaceutics at the University of Erlangen, Erlangen, Germany 91058, tel. +49 9131 85 29545, 

 works at the Ecolab Research Center in Eagan, Michigan. 

*To whom all correspondence should be addressed. Submitted: Jan. 23, 2008. Accepted. Apr. 23, 2008. 

What would you do differently?  Email your thoughts about this paper to 

1. J. Wechsler, "Modernizing Pharmaceutical Manufacturing," 

2. FDA/ORA, "Compliance Policy Guide, Sub Chapter 490.100," Process Validation Requirements for Drug Products and Active Pharmaceutical Ingredients Subject to Pre-Market Approval (CPG 7132c.08). 

3. M.J. Pikal and H.R. Costatino, "Lyophilization of Biopharmaceuticals" in 

, R.T. Borchard and C. R. Middaught, Eds. (AAPS Press, Arlington, VA, Vol. II, 2004).  

4. X. Tang, S.L. Nail, and M.J. Pikal, "Freeze-Drying Process Design by Manometric Temperature Measurement: Design of a Smart Freeze-Dryer," 

5.  H. Gieseler H, T. Kramer, and M.J. Pikal, "Use of Manometric Temperature Measurement (MTM) and SMART Freeze Dryer Technology for Development of an Optimized Freeze-Drying Cycle," 

6.  H. Gieseler, "Process Analytical Technology for Freeze-Drying: Cycle Optimization in the Laboratory," 

7.  F. Fonseca et al., "Collapse Temperature of Freeze-Dried Lactobacillus bulgaricus Suspensions and Protective Media," 

8.  E. Meister and H. Gieseler, "Evaluation of Collapse Temperatures by Freeze-Dry Microscopy: Impact of Excipient Concentration on Measured Transition and the Overall Dependence on Measurement Methodology," in Proceedings of Fifth World Meeting on Pharmaceutics and Pharmaceutical Technology (Geneva, Switzerland, March 2006). 

9.  S. Rambhatla et al., "Cake Shrinkage During Freeze Drying: A Combined Experimental and Theoretical Study," 

10. S. Tchessalov, "Principles of Lyophilization Cycle Scale Up" in   

Processing CPPR Freeze Drying of Pharmaceuticals and Biologicals Conference 

11.  M.J. Pikal, "Use of Laboratory Data in Freeze Drying Process Design: Heat and Mass Transfer Coefficients and the Computer Simulation of Freeze Drying,"

12. J.P Dolan, "Use of Volumetric Heating to Improve Heat Transfer During Vial Freeze Drying" in 

 (PhD Thesis, Virginia Polytechnic Institute, Blacksburg, VA, 1998). 

13. M.J. Pikal and S. Shah, "The Collapse Temperature in Freeze Drying: Dependence on Measurement Methodology and Rate of Water Removal from the Glassy Phase," 

Articles

Cycle design and robustness testing using advanced process analytical technology.

Peer-Reviewed Technical Note: Quality by Design in Freeze-Drying

Mannitol is popular as a crystalline bulking agent in freeze-drying, but tends to form different crystalline modifications which may negatively impact storage stability. Mannitol-based pharmaceutical formulations usually contain additional formulation additives which may alter the extent of crystallization and the modification of mannitol. In this study, the impact of pure sucrose, trehalose, polysorbate 80, and citric acid as well as multi-component systems of these additives, on mannitol crystallization was investigated. Pure mannitol and 10 formulations with excipients were lyophilized using identical freeze-drying conditions. The product cakes from center and edge vials were characterized using Karl Fischer residual-moisture analysis, X-ray powder diffraction, and differential scanning calorimetry. The study revealed substantial differences in residual moisture content and significant changes in crystallinity induced by even small amounts of additives, especially in the case of sucrose. 

Mannitol is the most commonly used bulking agent in freeze-drying formulation design. The benefit of using mannitol is that it crystallizes during freezing and permits drying processes at higher product temperatures, and thus with higher sublimation rates relative to purely amorphous systems (1). Mannitol, however, is known to form different crystalline modifications which compromises reproducibility of product characteristics and storage stability due to phase transformations (2, 3). The different modifications have been described extensively in the literature (4, 5). Because freeze-dried mannitol-based pharmaceuticals typically contain other excipients to fulfill the stabilization needs of the active pharmaceutical ingredient, the extent of crystallization and modification may be different than for pure mannitol (6). 

This study investigates the influence of frequently used additives on the crystallization of mannitol. The additives used were: sucrose and trehalose, both widely used as lyoprotectants to stabilize proteins or peptides during drying; polysorbate 80, a surfactant frequently added to avoid surface denaturation of proteins during freezing; and citric acid, a popular buffer system (1). 

All chemicals used were analytical grade and obtained from Sigma Chemical Company (Munich, Germany). Pure mannitol and 10 mixtures with excipients were lyophilized during a single run (see Table I). Nine 10-mL vials from Lutz GmbH (Wertheim,Germany) for each formulation were filled with 3-mL solution. Total solid content of each mixture was kept constant at 50 mg/mL. Freeze-drying was performed using a laboratory scale Lyostar II freeze-dryer (FTS Systems, Stone Ridge, NY). The lyophilization cycle  consisted of freezing at –40 °C, annealing at –15 °C for 3 h hours, and primary drying at –10° C and 100 mTorr (see Figure 1). Secondary drying was performed at 40 °C for 4 h. After the freeze-drying cycle, edge and center vials of each formulation were individually characterized using X-ray powder diffraction (XRPD) and differential scanning calorimetry (DSC) (7). The residual moisture (RM) for one vial of each formulation was determined by Karl Fischer analysis. 

. Residual moisture was determined by coulometric Karl-Fischer titration using a water vaporizer (Mitsubishi Chemical Company CA-06 with VA-06, Tokyo, Japan). Approximately 35–90 mg of the solid samples were transferred to the vaporizer unit where the sample was heated to 140° C. Extra dry nitrogen was used as a carrier to transfer the moisture into the measurement cell. 

A 30–50 mg sample was weighed into the XRPD sample holder, compacted in the sample block, and examined using an X'Pert MPD(Royal Philips, The Netherlands)with Cu K α radiation at 40kV/40mA and 25 °C. Scans were measured in the range 2θ = 0.5°–40° C with a step size of 0.02 °C. The diffractograms were plotted in Microcal Origin 7.5 (Originlab, Massachussettes), and the baseline corrected for a measurement offset.  

Table I: Binary and ternary mixtures used. 

The authors used a Mettler Toledo 822 DSC with STARe SW 9.01 software (Mettler Toledo, Switzerland) for thermal analysis. Approximately 20 mg of sample was weighed into a 40 μL aluminum pan and compressed in a dry-nitrogen atmosphere. Pans were then sealed hermetically and transferred into the DSC cell. Two different methods were applied. First, all formulations were scanned with a ramp rate of 10 °C/min from 0 °C–180 °C  to reveal potential glass transitions, melting events, or recrystallization. A lower ramp rate (2.5 °C/min) was used for some formulations showing inconclusive results at fast ramp rates.  

. As expected, the pure mannitol cake contained low levels of residual moisture, approximately 0.2% (w/w), due to its fullly crystalline character. Surprisingly, the binary mixtures of mannitol and polysorbate 80 showed even lower levels of moisture than Formulation 1; citric acid showed comparable moisture levels. Low levels of sucrose, however, led to substantially elevated RM results (e.g. 1.0% for Formulation 2 and 2.4% for Formulation 3). This trend can also be observed in the ternary mixtures 9 (1.7%) and 10 (2.0%). The amorphous phase consisting of a share of mannitol as well as sucrose retains higher concentrations of residual water. Here, the increased RM may be due to microcollapse of the amorphous phase onto the crystalline mannitol during primary drying, which restricts additional removal of water (8). The RM elevation can also be observed for trehalose, but to a lesser extent: both Formulation 4 and Formulation 11 contained 0.3% RM which is only slightly more than the pure mannitol cake. 

Table II: Summary of analytical results. 

The crystalline peaks in the diffractograms were compared to reference diffractograms of pure α-, β- and δ-mannitol and of mannitol hydrate reported in the literature (9-11). Note that the modification ratio was determined semi-quantitatively. Center and edge vials of each formulation showed the same qualitative composition for all formulations, excluding radiation effects and drying variations as possible factors for modification differences. The mannitol used for the solutions consisted of pure δ-modification. In contrast, practically all of the lyophilized formulations showed one main modification and some amount of one or more other modifications. Formulation 1 consisted of mostly β-mannitol and showed trace amounts of α- and δ-mannitol (see Figure 2). Similar compositions with higher amounts of α-modification were observed in formulations containing mannitol and citric acid (see Table I: Formulations 7 and 8). 

The addition of sucrose or trehalose led to an increased amorphous fraction of the cake and also affected the crystalline modifications. Mixtures with 0.5% and 1% sucrose showed mostly δ-mannitol and some α- and β-mannitol, but also contained significant amounts of mannitol hydrate, which corresponded well with the increased moisture content (see Figure 3). In Formulation 4 and Formulations 9–11, the δ- modification was dominant; the formulations containing sucrose also showed peaks of mannitol hydrate. Formulation 11 also contained some mannitol hydrate, which indicates effects of the increased amorphous fraction. Note that it was not possible to detect crystalline citric acid which might have remained amorphous during freeze-drying. 

The thermal analysis did not allow quantitative determination of mannitol modifications, but it was possible to obtain valuable information. In the majority of the formulations, the amorphous phase consisted mainly of sucrose, trehalose, or polysorbate 80and showed only one glass transition (Tg). The crystallization peaks gave an indication of the amount of mannitol that remained amorphous during lyophilization. Formulations with high moisture content displayed an additional small endothermic event at 130° C followed by an exotherm which could be caused by conversion of mannitol hydrate to an anhydrous modification. The melting point of pure α- and β-mannitol have been described as 166 °C and 166.5 °C, which clearly makes a separation impossible(12). Reports for the melting point of δ-mannitol vary from 150–158 °C, which is sufficiently lower than the others to allow identification (5). In the thermograms of the formulations studied, there were often smaller melting peaks of δ-mannitol followed by recrystallization and the melting peak of α- and β-mannitol which could be isolated using the slower heating rate. A sample thermogram is shown in Figure 4. 

The authors were able to lyophilize solutions of pure mannitol as well as various combinations of mannitol with excipients. The product cakes were characterized regarding residual moisture, crystallinity, and thermal characteristics. The excipients caused significant differences in physical properties, especially in the case of sucrose. Further investigation is required to identify patterns and consequences of these influences, in particular with an active pharmaceutical ingredient (protein or peptide). This study, however, clearly indicates that the effects of additives on crystallization of mannitol must be taken into account during formulation development of freeze-dried products. 

work in the Department of Pharmaceutics at the University of Erlangen, Erlangen, Germany 91058. tel. +49 9131 85 29545, 

*To whom all correspondence should be addressed. 

Submitted: Jan. 23, 2008. Accepted: Feb. 1, 2008.

Encyclopedia of Pharmaceutical Technology

 (Marcel Dekker, New York, 2002), pp. 1299–1326. 

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 10. L. Yu et al., "Existence of a Mannitol Hydrate During Freeze-Drying and Practical Implications," 

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Mannitol is the most commonly used bulking agent in freeze-drying formulation design. The benefit of using mannitol is that it crystallizes during freezing and permits drying processes at higher product temperatures, and thus with higher sublimation rates relative to purely amorphous systems (1). Mannitol, however, is known to form different crystalline modifications which compromises reproducibility of product characteristics and storage stability due to phase transformations (2, 3).