Importance of Using Complementary Process Analyzers for the

Anal. Chem. 2009, 81, 7639–7649

Importance of Using Complementary Process Analyzers for the Process Monitoring, Analysis, and Understanding of Freeze Drying T. R. M. De Beer,*,† M. Wiggenhorn,‡ R. Veillon,§ C. Debacq,§ Y. Mayeresse,§ B. Moreau,§ A. Burggraeve,† T. Quinten,| W. Friess,‡ G. Winter,‡ C. Vervaet,| J. P. Remon,| and W. R. G. Baeyens† Laboratory of Drug Analysis, Department of Pharmaceutical Analysis, Ghent University, Harelbekestraat 72, B-9000 Gent, Belgium, Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Ludwig-Maximilians-University, Butenandtstrasse 5-Building B, D-81377 Munich, Germany, Freeze Drying Department, GSK Biologicals, Rue de l’Institut 89, B-1330 Rixensart, Belgium, and Laboratory of Pharmaceutical Technology, Department of Pharmaceutics, Ghent University, Harelbekestraat 72, B-9000 Gent, Belgium The aim of the present paper is to demonstrate the importance of using complementary process analyzers (PAT tools) for the process monitoring, analysis, and understanding of freeze drying. A mannitol solution was used as a model system. Raman spectroscopic, nearinfrared (NIR) spectroscopic, plasma emission spectroscopic, and wireless temperature measurements (TEMPRIS) were simultaneously performed in-line and realtimeduringeachfreeze-dryingexperiment.Thecombination of these four process analyzers to monitor a freeze-drying process is unique. The Raman and NIR data were analyzed using principal component analysis (PCA) and multivariate curve resolution (MCR), while the plasma emission spectroscopic and wireless temperature measurement data were analyzed using univariate data analysis. It was shown that the considered process analyzers do not only complement but also mutually confirm each other with respect to process step end points, physical phenomena occurring during freeze drying (process understanding), and product characterization (solid state). Furthermore and most important, the combined use of the process analyzers helped to identify flaws in previous studies in which these process analyzers were studied individually. Process analyzers might wrongly indicate that some process steps are fulfilled. Finally, combining the studied process analyzers also showed that more information per process analyzer can be obtained than previously described. A combination of Raman and plasma emission spectroscopy seems favorable for the monitoring of nearly all critical freeze-drying process aspects. The overall aim of the present paper is to demonstrate the importance of implementing complementary process analyzers (PAT tools) in process analytical technology based production * Corresponding author. Tel.: +32-9-2648099. Fax: +32-9-2648196. E-mail: [email protected]. † Department of Pharmaceutical Analysis, Ghent University. ‡ Ludwig-Maximilians-University. § GSK Biologicals. | Department of Pharmaceutics, Ghent University. 10.1021/ac9010414 CCC: $40.75  2009 American Chemical Society Published on Web 08/14/2009

processes. Freeze drying of a mannitol solution was used as a model process for which Raman, near-infrared (NIR), and plasma emission spectroscopy and wireless temperature sensors were simultaneously applied as process analyzers. Process analytical technology (PAT) is a concept, proposed by the U.S. Food and Drug Administration (FDA) in 2002, which is expected to lie at the basis of the pharmaceutical “Good Manufacturing Practice” rules for the 21st century. By means of scientific, risk-based PAT frameworks, it is aimed to design and develop continuously monitored and controlled (by timely in-line, on-line or at-line measurements of the critical intermediate steps and end points during the process), well understood, and efficient processes that will consistently ensure a predefined quality at the end of the manufacturing process. To fulfill the PAT objectives in a process, it is necessary to apply a suitable combination of PAT tools (process analyzers, multivariate data analysis tools, end point monitoring tools, and knowledge management tools).1 PAT fits well in the paradigm shift in pharmaceutical manufacturing from a quality-by-design (QbD) perspective, as discussed in the regulatory guidelines (ICH Q8, Q9, and Q10). Freeze drying is a low-temperature drying process, based on the principles of heat and mass transfer, employed to convert aqueous solutions of (heat-)labile materials into solids with sufficient stability for distribution and storage. A lyophilization process starts with a freezing phase, where most of the water is converted into ice and the solutes are crystallized or transformed into a solid amorphous system. Therefore, the shelf temperature is adjusted to ensure that the product is cooled below the glass transition temperature or eutectic point. Next, a primary drying step is induced, where the ice crystals are removed under vacuum by sublimation. Herewith, the temperature is increased (but kept below collapse temperature) to supply heat for sublimation. The sublimated ice is driven to the condenser where the vapor pressure is lower than in the freeze-dryer chamber. The process ends with a secondary drying step under deep vacuum where most of the unfrozen water, i.e., water dissolved in the solid amorphous (1) http://www.fda.gov/cder/OPS/PAT.html.

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phase, is removed by diffusion and desorption.2 In some cases, secondary drying aims at removing hydrate water. It is clear that freeze drying is a complex and time-consuming multiple-step process during which the starting material (solution) undergoes several transformations leading to the end product (dry cake). Until now, several process analyzers have been developed (and used in industry) allowing the in-line and real-time determination of the freeze-drying step end points (e.g., thermocouples, manometric temperature measurements, vapor pressure method, etc.). These systems are mainly based on the continuous monitoring of the product temperature, the water vapor content, or pressure changes inside the freeze-dryer chamber. As long as water vapor can be observed in the freeze-dryer chamber, the ice sublimation (primary drying) and/or water desorption (secondary drying) processes are not finished. Product temperature monitoring allows determination of the ice and product crystallization (crystallization is an exothermic process resulting in product temperature increase), the primary drying end point (the product temperature equals to the shelf temperature when sublimation is finished; before this end point, the energy supplied by the shelves is used for sublimation), and the secondary drying end point. However, these techniques are mostly indirect and often disturb the normal freeze-drying procedure. They do not allow monitoring of all critical process aspects, do not improve understanding of the product behavior during the process, and/or cannot characterize the intermediate and end product quality parameters (solid state, polymorphic or hydrate/anhydrate transformations, processinduced transformations, residual moisture content, etc.), which are essential for real-time product release. Especially, contact of the process analyzers with the product should be avoided as this can influence the physical and chemical properties of the freezedried product, making the monitored vial unrepresentative for the unmonitored vials in the freeze dryer. Recently, new process analyzers have been proposed to overcome (some of) the shortcomings of these classically applied process monitoring tools. In-line and noninvasive Raman and NIR spectroscopic monitoring of products during freeze drying provides information about process step end points and product behavior and characteristics simultaneously. Our research group and Romero-Torres et al. described the in-line Raman spectroscopic monitoring of a freeze-drying process,3,4 while Bru¨lls et al. applied in situ and invasive NIR spectroscopy.5 Furthermore, we also outlined in a recently published study the complementary properties of Raman and NIR spectroscopy for freeze-drying monitoring by implementing both techniques simultaneously inside the freeze dryer.6 The critical freeze-drying aspects which can be monitored by both spectroscopic techniques and their (2) Pikal, M. J. In Encyclopedia of Pharmaceutical Technology; Swarbrick, J., Ed.; Marcel Dekker: New York, 2002; pp 1807-1833. (3) De Beer, T. R. M.; Allesø, M.; Goethals, F.; Coppens, A.; Vander Heyden, Y.; Lopez De Diego, H.; Rantanen, J.; Verpoort, F.; Vervaet, C.; Remon, J. P.; Baeyens, W. R. G. Anal. Chem. 2007, 79, 7992–8003. (4) Romero-Torres, S.; Wikstro ¨m, H.; Grant, E. R.; Taylor, L. S. PDA J. Pharm. Sci. Technol. 2007, 61, 131–145. (5) Bru ¨ lls, M.; Folestad, S.; Spare´n, A.; Rasmuson, A. Pharm. Res. 2003, 20, 494–499. (6) De Beer, T. R. M.; Vercruysse, P.; Burggraeve, A.; Quinten, T.; Ouyang, J.; Zhang, X.; Vervaet, C.; Remon, J. P.; Baeyens, W. R. G. J. Pharm. Sci. 2009, in press (DOI: 10.1002/jps. 21633). (7) Mayeresse, Y.; Veillon, R.; Sibille, P.; Nomine´, C. PDA J. Pharm. Sci. Technol. 2007, 61, 160–174.

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major advantages and disadvantages are mentioned in Table 1. Mayeresse et al. proposed the newly designed cold plasma ionization device to continuously monitor in-line and real-time the freeze-drying chamber moisture content, hence allowing the end point determination of the drying steps.7 The capabilities of this technique and its advantages and disadvantages are summarized in Table 1. Wiggenhorn et al. and Schneid and Gieseler described the use of wireless temperature sensors (TEMPRIS) for in-line product temperature monitoring and demonstrated their advantages over classically used thermocouples.8,9 On the basis of these papers, the wireless temperature sensors are also being evaluated in Table 1. Table 1 indicates that a combination of these process analyzers might allow freeze-drying monitoring of all critical process aspects. Therefore, the specific aims of the present study were • To combine these four recently introduced tools, each based on a different principle, for the simultaneous in-line monitoring of a freeze-drying process; hence, evaluating if they allow monitoring of all critical process aspects. To our best knowledge, the simultaneous implementation of these tools in a freeze-drying process and the focus on the importance of combining process analyzers is not yet described in literature • To evaluate their complementary properties • To determine if the combined use of these process analyzers provides more information than previously described • To examine whether a combined use of these process analyzers confirms previously described conclusions when these process analyzers were studied individually • To examine whether a combined use of process analyzers increases process understanding • To find the best combination of process analyzers allowing process monitoring of all critical process aspects. It is evident that this study is of major importance for the monitoring and analysis of freeze drying and that the main message, i.e., the importance of implementing complementary process analyzers, should be extrapolated to various processes. MATERIALS AND METHODS Materials. A 4 mL 5% (w/v) D-mannitol (further abbreviated as “mannitol”) solution was used as model for freeze drying. Mannitol is a widely used excipient in freeze-dried pharmaceutical formulations. As mannitol has a strong tendency to crystallize during freeze drying, it is mainly used as a bulking agent, resulting in elegant solid freeze-dried cakes.10 During lyophilization, mannitol can crystallize as anhydrous R, β, or δ forms or as mannitol hemihydrate.3,4 β-Mannitol and δ-mannitol were purchased from VWR International (Fontenay sous Bois, France) and VWR International (Poole, England), respectively. R-Mannitol was prepared by recrystallization as described by Burger et al.11 Mannitol hemihydrate was obtained by freeze drying as described in ref 3. Experimental Setup and Process Description. All freezedrying experiments were performed in an Amsco FINN-AQUA (8) Wiggenhorn, M. Evaluation of Advanced Approaches to Monitor Product Temperature during Lyophilization. Freeze Drying of Pharmaceuticals and Biologicals, Breckenridge, CO, August 7-9, 2008. (9) Schneid, S.; Gieseler, H. AAPS Pharm. Sci. Tech. 2008, 9, 729–739. (10) Pyne, A.; Surana, R.; Suryanarayanan, R. Pharm. Res. 2002, 19, 901–908. (11) Burger, A.; Henck, J. O.; Hetz, S.; Rollinger, J. M.; Weissnicht, A. A.; Sto¨ttner, H. J. Pharm. Sci. 2000, 89, 457–468.

Table 1. Overview of the Real-Time Process Monitoring Capabilities, Practical Skills, and Advantages and Disadvantages of Four Recently Proposed Freeze Drying Monitoring Tools, Each Individually Examineda Raman3,4,6 In-Line and Real-Time Detection of Critical Freeze-Drying Aspects residual moisture content intermediate product solid state + end product solid state + start water-to-ice conversion + end point water-to-ice conversion + start product crystallization + end point product crystallization + start primary drying + sublimation end point (end point primary drying) start secondary drying + end point secondary drying (desorption) ? end point removal hydrate water + annealing ? collapse + product temperature process fingerprint + Practical Skills, Advantages/Disadvantages steam sterilization possible easy to integrate reproducibility sensitivity (i.e., minimal load that could monitored) positioning tool scale-up possibilities global load monitoring automatic loading compatibility/stoppering devices aseptic handling noninvasive (no product contact) nondestructive heat conduction effects

? very good 1 probe/vial top vial ± ? ? + + ?

NIR5,6

TEMPRIS8,9

plasma emission spectroscopy7

+ + + + + + + + + + ? + +

? ? ? ? + + + + ? ? + + +

+ + + + ? +

? very good 1 probe/vial side wall ± ? ? + + ?

+ very good 1 sensor/vial center bottom ± ? ? ? + ?

+ + very good up to 1 vial (0.5 mL) mounted to chamber top + + + + + + ? -

a (+) indicates the ability to monitor the considered process aspect, while (-) means the non-applicability of the tool. (?) indicates that the considered process aspect or analyzer skill is not evaluated in the reference paper and that it cannot be derived from the conclusions in the reference papers.

Figure 1. Experimental setup.

GT4 freeze dryer (GEA, Ko¨ln, Germany). For the in-line and realtime spectroscopic monitoring, the plasma ionization device probe was mounted directly on top of the freeze-dryer chamber (Figure 1). Raman and NIR noncontact probes were built into the freezedryer chamber (Figure 1). Noncontact probes are required, as contact of measurement tools with the freeze-dried product influences the process, making the monitored vials unrepresentative for the other vials.3,4,6 The fiber-optic cables connecting the

Raman and NIR probes to the respective spectrometers were guided through the door of the freeze-dryer chamber. The Raman probe, positioned above the vial, and the NIR probe, placed in front of the sidewall at the bottom of the vial (i.e., NIR measurements done through the glass vial in contrast to the Raman measurements), were each focused on a different vial for reasons described in our previous paper.6 Focusing both probes on the same vial results in saturated Raman spectra, as the nonabsorbed Analytical Chemistry, Vol. 81, No. 18, September 15, 2009

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Table 2. Freeze Drying Conditions for Experiment 1 process phase freezing

primary drying

time (min)

shelf temperature (°C)

pressure (mbar)

0 5 25 85 145 165 175–2500

20 2 –5 –30 –45 –45 –20

1000 1000 1000 1000 1000 1000 0.8–1.0

NIR light is reflected to the Raman probe, hence being collected together with the Raman scattering.6 The position of the Raman probe (top) and NIR probe (side wall) to the monitored vials is also discussed in ref 6. Up to three wireless temperature sensors were used to monitor product temperature during freeze drying (Figure 1). One sensor was placed just above the solution to freeze-dry (to avoid contact), while another sensor was placed just below the surface of the freeze-dried solution. A third sensor was completely immersed in the solution (bottom center) as described in ref.9 In this paper, Schneid and Gieseler placed the temperature sensor at the bottom center of the vial, as the ice sublimation process finishes at this location in the vial. The freeze-drying conditions applied for the first experiment are listed in Table 2. On the basis of the obtained results and conclusions for this experiment, new experimental conditions were set up. New experiments were, thus, planned on the basis of the results from previous experiments (see Results and Discussion section). An overview of all performed experiments and their settings is given in Table 3. Measurement Tools. Raman Spectroscopy. A RamanRxn1 spectrometer (Kaiser Optical Systems, Ann Arbor, MI) equipped with an air-cooled charge coupled device (CCD) detector (backilluminated deep depletion design) was used in combination with a fiber-optic noncontact probe to monitor the lyophilization processes in-line and noninvasively. As the Raman probe was directly focused on the product to freeze-dry, the glass vial did not interfere with the Raman signal. The laser wavelength during the experiments was the 785 nm line from a 785 nm Invictus NIR diode laser. All spectra were recorded at a resolution of 4 cm-1 using a laser power of 400 mW. Data collection, data transfer, and data analysis were automated using the HoloGRAMS data collection software (Kaiser Optical Systems), the HoloREACT reaction analysis and profiling software (Kaiser Optical Systems), the Matlab software (The Mathworks; version 7.7), and the Grams/AI-PLSplusIQ software (Thermo Scientific; version 7.02). Thirty two second exposures were used for in-line monitoring of freeze drying. Spectra were collected every minute. Spectra were preprocessed by baseline correction (Pearson’s method) before data analysis. NIR Spectroscopy. Diffuse reflectance NIR spectra were continuously collected in-line and noninvasively during freeze drying using a Fourier-Transform NIR spectrometer (Thermo Fisher Scientific, Nicolet Antaris II near-IR analyzer) equipped with an InGaAS detector, a quartz halogen lamp, and a fiber-optic noncontact probe, which was placed next to the vial. Data analysis was done using Thermo Fisher Scientific’s Result software, SIMCA-P (Umetrics, version 11), and Matlab (The Mathworks, version 7.7). Each spectrum was collected in the 10 000-4000 7642


cm-1 region with a resolution of 16 cm-1 and averaged over 32 scans. All spectra were preprocessed using standard normal variate transformation (SNV) and mean centered before data analysis. Plasma Emission Spectroscopy. The applied cold plasma ionization device (Lyotrack 100, Adixen, France) is an innovative tool, based on the Inductive Coupled Plasma/Optical Emission Spectroscopy (ICP/OES) technique, allowing in-line and real-time water vapor determination in the freeze-dryer chamber during drying.7 The device is composed of a quartz tube which directly contacts the lyophilization chamber and is connected directly to the freeze-dryer ports via an ISO-KF25 or triclamp flange on top of the freeze-dryer chamber. All parts of the device in contact with the internal freeze-dryer atmosphere can withstand sterilization conditions. As water vapor is led to the condenser, the Lyotrack system is supposed to be able to detect the primary and secondary drying end points. Two gases are mainly present in a freeze dryer during drying: water vapor coming from the vials and nitrogen coming from the pressure regulation. The pressure during drying in the freeze dryer is air-regulated, air consisting of about 80% nitrogen. The radiofrequency source (440 MHz) from the plasma ionization device creates a cold plasma in the quartz tube under vacuum (i.e., < 3 mbar). The electrons from the gas atoms and molecules in the freeze dryer are moved to a discrete higher energy state by absorbing this radiofrequency energy. The electrons spontaneously return to their normal state and release this energy by emitting a photon. The wavelength of these photons is characteristic for each molecule and atom. The emitted photons are collected with an optical fiber and diffracted with an optical spectrometer. The optical spectrum is analyzed by the plasma sensor software and the humidity curve, varying between 0 (no water vapor) and 1 (saturated with water vapor), and is displayed in real-time. As water vapor and nitrogen are the two main gases in the freeze-dryer chamber, the Lyotrack system measures the ratio of water vapor to nitrogen (labeled as humidity in the software) during the drying steps. Wireless Temperature Measurements (TEMPRIS). The recently proposed wireless temperature sensors (Temperature Remote Interrogation System, TEMPRIS) were used as fourth innovative process analyzers for this study.8,9 The TEMPRIS system (IQ Mobil Solutions GmbH, Wolfratshausen, Germany) used in this study consisted of eight sensors, the interrogation unit, and a computer system with CarLog software (IQ Mobil Solutions GmbH, Wolfratshausen, Germany). The operation principle of TEMPRIS is well described in refs 8 and 9. The placement of the sensors in the vials is described in the Experimental Setup and Process Description. Solid State Characterization. For each monitored freeze-drying process, the solid state of the end product was identified by comparing the Raman and NIR spectra of the product with the reference spectra of the four crystalline mannitol forms, as described in our previous studies (mainly visual inspection of the Raman spectra).6 The mannitol solid state in the freeze-dried end products was further determined and confirmed via X-ray powder diffractometry (XRPD). Process Analysis. As four process analyzers were applied to continuously collect data during the freeze-drying processes, a

Table 3. Overview of Experimental Conditions for All Performed Freeze Drying Experiments experiment number 1 2 3 4 5

WTC adapted freeze-drying conditions compared to Table 2 primary drying at -15 °C freezing between 25 and 120 min from -5 to -45 °C primary drying at -15 °C until 1920 min secondary drying at 40 °C until 2500 min idem as experiment 3 7% mannitol solution 3.5 mL filled vial

Raman

NIR

plasma

above

just inside

immersed

yes (vial 1) yes (vial 1) yes (vial 1)

yes (vial 2) yes (vial 2) yes (vial 2)

yes yes yes

no no no

no no no

yes (vial 3) yes (vial 3) yes (vials 1 and 2)

yes (vial 1) yes (vial 1)

yes (vial 2) no

yes yes

no yes (vial 4)

no yes (vial 3)

yes (vials 3 and 4) yes (vial 2)

huge amount of complex data was obtained per process. As 1 Raman spectrum and 1 NIR spectrum were obtained per minute during the processes, which sometimes took over 30 h, chemometric tools were necessary to extract useful information from the large data sets. The plasma and temperature data were analyzed univariately, as only one data point per process time point was obtained. The Raman and NIR spectra collected per monitored freezedrying process were each introduced into a data matrix (D), resulting in an NIR data matrix and a Raman data matrix per process. Each D was analyzed using principal component analysis (PCA) and/or multivariate curve resolution (MCR). A mathematical explanation of these chemometric techniques can be found in our previous studies, for which also PCA and MCR were applied for data analysis of NIR and Raman spectra collected during freeze drying.3,6 RESULTS AND DISCUSSION Freeze-Drying Monitoring Using Four Different Process Analyzers Simultaneously. In a first experiment, three vials were freeze-dried (Tables 2 and 3). One vial was monitored using Raman spectroscopy, a second one using NIR spectroscopy, and a wireless temperature sensor was completely immersed at the bottom center of the third vial (Table 3). Our previous studies showed that the Raman spectral differences between the different mannitol solid states (R, β, δ, and hemihydrate) can be clearly distinguished in the 1000-1170 cm-1 spectral range. Furthermore, several critical process aspects and several phenomena occurring during freeze drying can be evaluated by monitoring in this region. Since ice produces a Raman signal at 215 cm-1, the intensity of this band was also monitored during freezing (ice formation) and primary drying (sublimation).3,6 As water and ice produce no signal at 1000-1170 cm-1, all visible Raman bands in this spectral range originate from mannitol. NIR spectra were analyzed between 4466 and 7243 cm-1 as ice produces a broad absorption signal in this region. The different crystalline mannitol forms can be distinguished via NIR in the 4330-4450 cm-1 spectral range.6 For easy data analysis and interpretation, the spectroscopic data obtained during the freezing step and during the drying steps were analyzed separately. Figure 2a shows the shelf temperature and chamber pressure from the first 300 process minutes, combined with the product temperature (vial 3) measured by the wireless temperature sensor. The product temperature profile mainly followed the shelf temperature profile during the freezing step, although with an upward shift of approximately 8 °C. This shift is caused by the different

heat transfer resistances between the inner of the shelf and freezedried product, being the shelf itself, the glass vial, and the frozen product. After 63 min, a sudden temperature increase occurred, indicating the exothermic ice crystallization. After 86 min, the product temperature dropped exponential-like to approximately 8 °C above shelf temperature indicating that the ice formation was completed.5 Between approximately 105 and 140 min, the product temperature curve shows a broad plateau (however, the plateau tilt moving downward), most likely indicating mannitol crystallization. This is hardly visible and does not allow clear detection of the start and end of mannitol crystallization. However, when solutions containing higher mannitol concentrations (e.g., 10%) are freeze-dried, this plateau is better visible (Figure S-1; see the Supporting Information). After 165 min, primary drying started. The small product temperature decrease occurring at the drying start (165 min) (Figure 2a) results from the fact that the frozen solution adopted the equilibrium temperature that cor-

Figure 2. (a) Process settings (shelf temperature and chamber pressure) from the first 300 process minutes, combined with the measured product temperature (vial 3) using the wireless temperature sensor (experiment 1). (b) Process settings and measured product temperature during experiment 1. Analytical Chemistry, Vol. 81, No. 18, September 15, 2009

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responded to the prevailing water vapor pressure above the ice surface.5 The Goff-Gratch equation allows one to calculate the temperature required at a certain pressure for ice sublimation to occur12

(

log p ) -9.09718

)

( ) (

T0 T0 - 1 -3.56654 log + T T 0.876793 1 -

)

T + log p0 T0

where p is the saturation water vapor pressure over ice (hPa), T is the air temperature (K), T0 is the ice-point (triple point) temperature (273.16 K), and p0 is p at the ice-point pressure (6.1173 hPa). The product temperature at the primary drying start is -33 °C (Figure 2a). On the basis of the Goff-Gratch equation, it can be derived that a pressure of 0.28 mbar is required to have ice sublimation at this product temperature. However, as primary drying was set at 1.00 mbar and as it takes some time before this chamber pressure is reached, the product temperature decrease between 165 and 174 min cannot come from sublimation. The used freeze-drying system does not allow monitoring the true shelf temperature and chamber pressure during processing. It is reasonable that this temperature decrease from -33 to -35 °C is caused by the significant drop of pressure from 1000 mbar to approximately 3 mbar between 165 and 174 min. At the primary drying start, the shelf temperature is programmed to increase from -45 to -20 °C. However, it always takes some time before this shelf temperature increase is fulfilled. From the Goff-Gratch equation, it can be derived that a product temperature of -21 °C is required at 1 mbar for ice sublimation to occur. Therefore, the product temperature increases between 174 and 214 min by the heat supplied by the shelves until the pressure is below the vapor pressure of ice. After 214 min, a product temperature of -18 °C is reached, enabling ice sublimation. The small temperature decrease after 249 min can be indicative for the end of an exothermic process. An explanation can be mannitol crystallization during primary drying as mannitol is known to crystallize only partly during the freezing step.13 Hawe et al. described the partial crystallization during cooling of a 5% mannitol solution, with further crystallization at -19.3 °C during rewarming of the sample.13 The mannitol crystallization in vial 3, hence, appears to be finished after 249 min. A second possible reason for the temperature decrease after 249 min is that the shelf temperature increased so slowly that the temperature for sublimation (being an endothermic process, hence causing a possible product temperature decrease) to occur at 1mbar was only reached after 249 min. Monitoring of the true shelf temperature and chamber pressure is necessary to clearly elucidate the product temperature changes occurring at the primary drying start. Figure 2b shows the process settings and measured product temperature during the whole process. After 250 min, the product temperature slightly increases by the mass transfer resistance in the dry product layer formed on the surface of the frozen solution, which grew as the drying process proceeded. After approximately 1553 min, far after the start of the drying step, the product temperature suddenly (12) Wexler, A. J. Res. Nat. Bur. Stand. 1977, 81, 38–44. (13) Hawe, A.; Friess, W. Eur. J. Pharm. Biopharm. 2006, 64, 316–325.

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increases faster, indicating that sublimation reached the area around the temperature sensor which was placed at the bottom center of the vial. Ice sublimation generally proceeds from the top to the bottom and to a lesser extent, from the side to the center of the vial.14 Figure 2b clearly shows a constant product temperature after 2356 min, which is supposed to correspond to the drying end point in vial 3. The commonly accepted definition of reporting the end point of primary drying is when the temperature reading of the sensor is essentially equivalent to or higher than the shelf temperature and is constant over time. A summary of the temperature sensor observations for experiment 1 is given in Table 4. The Raman data analysis and interpretation are shown in Figures 3 and S-2 (see the Supporting Information), and the observations are reported in Table 4. Extensive textual outline of Figures 3 and S-2 (see the Supporting Information) is not given, as this was done in our previous manuscripts.3,6 The first 77 collected Raman spectra are clustered in the scores plot (Figure 3) indicating that no solidification occurs during the first 77 process minutes. After 78 min, the spectral scores evolve to a second cluster in the scores plot reached after 103 min, indicating that water to ice conversion started after 87 min and ended after 103 min. After 113 min, the spectral scores move to a third cluster, representing the mannitol crystallization process, which finished after 133 min. After starting the primary drying step, the only spectral variation is peak intensity increase of the Raman bands due to the product temperature increase (the Raman signal is temperature dependent) and due to the further mannitol crystallization. The Raman spectra did not change anymore after 250 min (data not shown). Figure S-2 (see the Supporting Information) plots the peak intensity of the 215 cm-1 Raman band, representing ice, versus process time. This plot confirms that ice crystallization started after 78 min and finished after 103 min. Furthermore, Figure S-2 (see the Supporting Information) shows that ice sublimation is finished after 207 min at the measuring location of the Raman probe. However, it should be emphasized that this is not representative for the drying end point of the whole vial, as Raman measurements are done at the surface of the freeze-dried solution, while sublimation finishes at the bottom center of the vial. The NIR data analysis and interpretation is done as in ref 6 and summarized in Figures S-3a and S-3b (see the Supporting Information). The conclusions are listed in Table 4. For the freezing step data, the loadings of the first PC, capturing 98.1% of the total spectral variance, correspond to the spectrum of ice (data not shown). In Figure S-3a (see the Supporting Information), the scores for the first PC were plotted versus process time and indicated that ice crystallization started after 80 min and ended after approximately 101 min. After 101 min, the NIR spectra changed immediately, slightly further until 136 min (Figure S-3b in the Supporting Information). This confirms our previous findings described in ref6 that mannitol crystallization immediately started after ice crystallization finished (or even starts during ice crystallization) and that several phenomena during freeze drying are probably not uniformly occurring throughout the whole vial. The illumination of a large part of the vial (where phenomena (14) von Graberg, S.; Gieseler, H. Proc. AAPS Annual Meeting and Exposition, San Diego, CA, 2007.

Table 4. Observations (min) for All Performed Freeze Drying Experimentsa experiment number 1

2

3

4

5

a

plasma

WTC

observation

Raman

NIR

vial 1

vial 2

vial 3

vial number start water-to-ice conversion end ice crystallization start mannitol crystallization end mannitol crystallization start primary drying end mannitol crystallization during drying end primary drying solid state

1 78 103 113 133 166 250 207 β + HH

2 80 101

X

X

X

3 63 86

190

190

190

2083 β + HH

2112/2150

2112/2150

2364

165 249 2356


1 87 107 122 128 165 248 232 HH

2 82 103

X

X

X

3 57 83

200

200

200

800 β + HH

1554/1658

1554/1658

1872 1872

165 250 1860

vial number start water-to-ice conversion end ice crystallization start mannitol crystallization end mannitol crystallization start primary drying end mannitol crystallization during drying end primary drying start secondary drying end secondary drying solid state before sec. drying solid state after sec. drying

1 105 139 152 163 170 207 245 1936 2209 β + HH R

2 106 135

X

X

X

216

216

1469 1927 2167 β + HH R

1676/1848 1930 2148/2244

1676/1848 1930 2148/2244

vial number start water-to-ice conversion end ice crystallization start mannitol crystallization end mannitol crystallization start primary drying end mannitol crystallization during drying end primary drying start secondary drying end secondary drying solid state before secondary drying solid state after secondary drying

1 121 145 157 not 170 277 255 1828 2207 β + HH R

2 117 155

1/2

1/2

3/4

not 648

not 186

186

889 1915 2218 β + HH R

1502/1742 1934 2180/2200


1 74 91 104 118 145 199 303 β + HH

136 1030

136 632

not 832

vial 4

above

just inside

immersed

1 105 138

2 106 132

168 218 1668 1927 2300

168 218 1834 1927 2300

3/4

3 97 124

4 96 124

186

186

1502/1742 1934 2180/2200

1858 1934 2290

1858 1934 2290

166 216 1851 1924 2200

166 216 1841 1924 2200

1

2

3

4

4 66 88

3 65 84

2 61 81

153

153

153

153

1056

1868

1800

1738

148 205 1220

148 200 1332

148 205 1579

HH ) mannitol hemihydrate.

are not occurring uniformly) by the NIR light source (large spot size) explains why an immediate transition between the start of mannitol crystallization and the end of water to ice conversion was, thus, observed. After 136 min, the spectra remain constant, indicating that mannitol crystallization was terminated (Figure S-3b in the Supporting Information). After performing PCA on the NIR spectra collected during drying, the scores and loadings of the first PC, capturing 99.1% of the total spectral variance, were studied. The loadings of the first PC suggested variation in the spectra due to changes of the absorption bands of ice and crystalline mannitol (data not shown). The scores of PC 1 versus process time plot let us conclude that as drying progressed, ice was sublimated and only mannitol was detected (data not shown). Presumably, drying started after 1030 min and finished after 2083 min. In a last step, PCA was performed on the NIR spectra

collected during the first 400 drying minutes. This was done to clarify the Raman and TEMPRIS findings obtained shortly after introduction of the vacuum. However, the scores of the first PC versus process time plot shows that the vacuum introduction and shelf temperature change do not affect the NIR spectra (data not shown). The spectra did not show any change during the freezing to drying step transition. The plasma profile obtained during experiment 1 is shown in Figure 4. As the vacuum was introduced at the primary drying, the plasma signal increased rapidly over 24 min (between 190 and 214 min) and reached almost vapor saturation inside the freeze-dryer chamber. The plasma signal decreased already 6 min later (due to the low number of vials and, hence, low amount of water vapor in freeze dryer) according to a sigmoid shaped curve. After 2364 min, the signal became stable indicating that most of Analytical Chemistry, Vol. 81, No. 18, September 15, 2009

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Figure 3. PC 1 versus PC 2 scores plot obtained after PCA from the first 300 in-line collected Raman spectra during experiment 1. PC 1 captures 79.81% of total spectral variance, while PC 2 captures 12.13%.

Figure 4. Cold plasma sensor response during experiment 1.

Figure 5. Measured product temperatures (TEMPRIS) during primary and secondary drying in the Raman and NIR monitored vials (experiment 3).

the vapor had been replaced by nitrogen and that the drying end point was reached. Furthermore, the curve shows bends after 2112 and 2150 min. Hence, the drying end point of the three freezedried vials can be individually determined from this plasma profile. The first two drying end points (2112 and 2150 min) most probably correspond to the Raman and NIR monitored vial. Which end point is corresponding to which vial is unclear as none of the Raman 7646


Figure 6. Plasma emission spectroscopy profile for experiment 3.

and NIR determined sublimation end points match the plasma conclusions. The last drying end point (2364 min) in the plasma profile most probably corresponds to the vial containing the temperature sensor (vial 3), as this value is close to the end point observed by the temperature sensor (2356 min). Furthermore, longer drying times were expected in the vial containing the completely immersed sensor, as the sensor itself takes up some volume in the vial, resulting in a thicker cake, which obviously requires longer drying times. A major advantage of the plasma system is that it is sensitive to determine the drying end point of a single vial. Hence, plasma emission spectroscopy allows one to determine when the last vial in a freeze dryer is dried. This is of major importance, as heterogeneity always occurs between the vials. Furthermore, Mayeresse et al. proved that the profile itself can be considered as a fingerprint for a freeze-drying process.7 Solid state analysis of the freeze-dried products was done as described in Solid State Characterization. In both the Raman and NIR vial, a mixture of mainly mannitol hemihydrate and β-mannitol was found (Table 4). On the basis of the observations for the first experiment, summarized in Table 4, it can be concluded that the freezing step progresses similarly in the Raman and NIR vial (vials 1 and 2). Evidently, there are minor intervial variations. The freezing process progress in vial 3 is different compared to vials 1 and 2,

as the ice crystallization occurs earlier probably because the temperature sensor is in contact with the product. It is well-known that invasively monitored vials are not representative for the vials containing no sensor due to variations in the nucleation and freezing behavior of the solution containing the sensor.2,15 The TEMPRIS drying end points match perfectly with the plasma emission spectroscopy, indicating that both tools are suitable for drying end point detection. However, the temperature sensor clearly affects the freezing step, which consecutively affects the drying steps. Data analysis and interpretation was performed similarly for all experiments in this study. To verify the general conclusions obtained from experiment 1, a second and similar experiment was performed, except that drying was performed at -15 °C (observations in Table 4). This second experiment resulted in similar observations and conclusions as for experiment 1. It is evident that shorter drying times were required as the shelf temperature during drying in the second experiment was higher than for the first experiment. Experiments 1 and 2 confirm the conclusions from3-6 about the benefits of Raman and NIR spectroscopy for freeze-drying monitoring purposes. Only the drying end point obtained from the NIR measurements does not correspond to the plasma data. Probably NIR underestimates the drying end point as the NIR probe is placed in front of the side wall of the monitored vial, while sublimation finishes at the bottom center of the vial. Furthermore, it is clear that vials containing a temperature sensor show a different freezing behavior compared to the Raman and NIR monitored vials. Moreover, as the temperature sensor takes some volume in the vial, a thicker freeze-dried cake is obtained, resulting in longer drying times. Plasma emission spectroscopy is an excellent tool for determination of the primary drying end point and can confirm the drying end points obtained from the TEMPRIS measurements. Even the drying end point in each individual vial can be detected. Secondary Drying. As described in refs 3 and 6 and confirmed in experiments 1 and 2, the freeze-drying process of a mannitol solution often results in a mixture of crystalline forms. However, mannitol hemihydrate is almost always formed. The presence of a hydrate should be avoided as this hydrate water might be released during storage and, hence, cause stability problems of the freeze-dried product. Mannitol hemihydrate can be removed during freeze drying by performing secondary drying at 40 °C.3,4 Therefore, a new experiment (experiment 3) was performed in which, following primary drying at -15 °C, the shelf temperature was elevated to 40 °C (Table 3). Also a slightly modified (slower) freezing program was used, as a low freezing rate stimulates the formation of mannitol hemihydrate.3 Furthermore, temperature sensors were placed in the vials monitored via Raman and NIR. This allowed one to find out whether the TEMPRIS observations could be confirmed by the Raman and NIR measurements. The obtained conclusions are listed in Table 4. The Raman and NIR observations correspond perfectly to the TEMPRIS measurements in their respective vials during the freezing step. When plasma emission spectroscopy is used, it is possible to detect the primary drying end points from the individual vials, although without the (15) Carpenter, J. F.; Pikal, M. J.; Chang, B. S.; Randolph, T. W. Pharm. Res. 1997, 14, 969–975.

Figure 7. Process settings and measured product temperatures (red ) vial 2; blue ) vial 3; green ) vial 4) during experiment 5.

ability to clearly identify the vials. The primary drying end points via plasma correspond well to the end points concluded from the TEMPRIS measurements (Table 4). Figure 5 shows the obtained TEMPRIS profile from experiment 3 during primary and secondary drying. Ice sublimation starts in the Raman vial around the sensor after 1039 min and after 1367 min in the NIR vial. Primary drying is finished after 1668 min in the Raman vial and after 1834 min in the NIR vial. In both vials, a major temperature increase is observed after 1927 min due to the onset of secondary drying. After approximately 2300 min, the product temperature in both vials reaches 35 °C (just below shelf temperature), which might indicate that secondary drying is finished. However, the secondary drying end point detection using the wireless temperature sensors is not as obvious as during primary drying end point detection. The primary drying end points correspond well to the plasma results (Figure 6, Table 4). The plasma profile further clearly shows the start of secondary drying and the end points of secondary drying for both vials. As secondary drying is used to transform the hydrate form of mannitol to an anhydrous form, this should be visible in the Raman and NIR spectra. Figure S-4a (see the Supporting Information) shows the Raman spectral changes between 1800 and 2300 min (i.e., the process period during which secondary drying is occurring according the plasma and wireless temperature couple (WTC) measurements). All mannitol is transformed into the R form. PCA (data not shown) was performed on these spectra to exactly determine when this transformation started and ended (i.e., start and end point of secondary drying, Table 4). The Raman conclusions approach roughly the plasma conclusions. However, the plasma tool appears to be a more exact tool for end point determination. Figure S-4b (see the Supporting Information) shows the second derivates of the NIR spectra collected between 1800 and 2300 min in the 5319-5050 cm-1 range. It was shown in previous studies that it is possible to distinguish between bound and surface water in this spectral range, as well as in the 7092-6756 cm-1 range (this range not shown).6,16,17 Surface water has peaks at 7002 and 5249 cm-1, while bound water has peaks at 6825 and 5136 cm-1. As secondary drying is performed under vacuum and at 40 °C, released bounded water is immediately transferred to (16) Cao, W.; Mao, C.; Chen, W.; Lin, H.; Krishnan, S.; Cauchon, N. J. Pharm. Sci. 2006, 95, 2077–2086. (17) Zhou, G. X.; Ge, Z.; Dorwart, J.; Izzo, B.; Kukura, J.; Bicker, G.; Wyvratt, J. J. Pharm. Sci. 2003, 92, 1058–1065.


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Table 5 Raman In-Line and Real-Time Detection of Critical Freeze-Drying Aspectsa residual moisture content intermediate product solid state + end product solid state + start water-to-ice conversion + end point water-to-ice conversion + start product crystallization + end point product crystallization + start primary drying + sublimation end point (end point primary drying) start secondary drying + end point secondary drying (desorption) ? end point removal hydrate water (b annealing ? collapse + product temperature process fingerprint +

NIR

TEMPRIS

plasma emission spectroscopy

-b + + + + + -b + + + ? + +

+b +b (b (b + + + ?b -b ? + + +

+ + + + +b +

a (+) indicates the ability to monitor the considered process aspect, while (-) means the non-applicability of the tool. (?) indicates that the considered process aspect or analyzer skill is not evaluated in the reference paper and that it cannot be derived from the conclusions in the reference papers. b Bold.

the freeze-dryer condenser. Hence, surface water was hardly observed in the NIR spectra during the process. Figure S-4b (see the Supporting Information) shows a decrease of the bound water signal during secondary drying. PCA (data not shown) was performed to determine the start and end point of the solid state transformation process (results in Table 4). Again, plasma emission spectroscopy appears to be a more exact tool for end point determination. A fourth experiment was done under the same freeze-drying conditions as for experiment 3 but with the two temperature sensors placed in independent vials (hence 4 vials in the freeze dryer, Table 3) to examine if the contact between the temperature sensors and the product influences the end product solid state. All observations for experiment 4 are listed in Table 4. Confirming previous observations, the vials containing the sensors show different (end point) observations compared to the NIR and Raman monitored vials, due to the contact of the sensors with the freezedried product. The observed solid states before the introduction of secondary drying in the Raman and NIR vials, each containing a temperature sensor, in experiment 3 is again a mixture of mainly mannitol hemihydrate with a little β-mannitol. For experiment 4, the Raman and NIR vial did not contain a sensor. Also here, a mixture of mainly mannitol hemihydrate and β-mannitol was observed. This indicates for this formulation that the contact from the temperature sensor with the product to freeze-dry did not influence the obtained solid state. Performing secondary drying at 40 °C always resulted in R-mannitol. It should be added for experiments 3 and 4 that the end point of mannitol crystallization during freezing was hardly visible as mannitol crystallization finished under these slow freezing conditions just before or just after the onset of drying. On the basis of the experiments described so far, plasma emission spectroscopy is the most accurate monitoring tool for the drying end point, while Raman spectroscopy is the best tool for monitoring of the freezing step. We proved that NIR spectroscopy is not suitable for drying monitoring (inaccurate end point detection) and that this technique does not provide all information during the freezing step. Therefore, NIR was excluded for the next experiment. Furthermore, it became clear that contact of the 7648


wireless temperature sensors with the product should be avoided. Therefore, an additional experiment was performed to determine how the temperature sensors allow process monitoring when they are placed just above the solution to freeze-dry. Examination of Temperature Sensor Position. For the last experiment (experiment 5), 4 vials were freeze-dried. The freezedrying conditions for this experiment were randomly chosen (Table 3). A first vial (vial 1) was monitored using Raman spectroscopy while the three remaining vials were monitored using temperature sensors. The temperature sensors were differently positioned in each vial. In vial 2, the temperature sensor was completely immersed in the solution to freeze-dry (similar to the previous experiments). In vial 3, only the tip of the sensor was immersed in the solution. Hence, the sensor was not in contact with the bottom center of the vial. In vial 4, the temperature sensor was placed just above the solution to freezedry (no contact between sensor and product). The Raman and TEMPRIS observations are shown in Table 4. The temperature profiles obtained from the three sensors of experiment 5 are shown in Figure 7. Also the sensors placed just below the surface of the solution and even above the product are able to detect the start and end of ice crystallization and the drying end point (Figure 7 and Table 4). Although the measured absolute temperatures are different, the temperature changes due to ice crystallization and drying end points are clearly visible in all cases, indicating the high sensitivity of the sensors. Drying is finished latest in the vial where the sensor is completely immersed (due to the increased cake thickness). Drying was fastest in the Raman monitored vial, even compared to the vials where the sensor is placed above the solution (i.e., without contact). This was confirmed by other experiments (data not shown). This indicates that the sensors might influence the product freeze drying, even when they are not in contact with the product. This is confirmed by the onset time of water to ice conversion. Ice crystallization starts first in the vial where the temperature sensor is completely immersed, due to the contact between sensor and solution. Ice crystallization also seems to start a bit faster in the vials having the sensors just inside or above the product. The presence of a stopper and sensor in a vial (even not in contact with the product)

might be the cause for more uncontrolled freezing than when they are not present. Another reason for faster drying in the Raman vial might be that the Raman vial has no stopper while the TEMPRIS monitored vial does (extra sublimation resistance). CONCLUSION This study proves the importance of using complementary process analyzers for freeze-drying monitoring. Four recently proposed PAT tools (Raman spectroscopy, NIR spectroscopy, plasma emission spectroscopy, and wireless temperature sensors) were simultaneously implemented to monitor freeze-drying processes. It was found that a combination of Raman and plasma emission spectroscopy allows the monitoring of nearly all critical process aspects. Raman spectroscopy can give insight into the product behavior, whereas plasma emission spectroscopy gives information about the drying process. Raman and NIR allow one to study the bound and/or surface water dynamics during secondary drying. Complementary process analyzers cannot only confirm but also complement each other. The unique combination of the four process analyzers applied for this study further helped to counter earlier described conclusions when these process analyzers were studied individually (summarized in Table 1), to find more information than previously described, and to increase freeze-drying process understanding. Table 5 is a re-evaluation

from the first part of Table 1. The changes compared to Table 1 are mentioned in bold. Finally, it became evident that complementary process analyzers, based on different principles, undoubtedly help to increase process understanding. The main message of this study, being the importance of implementing complementary process analyzers in PAT-based processes, should be extrapolated to other kinds of (pharmaceutical) processes. SUPPORTING INFORMATION AVAILABLE Figures S-1 to S-4 are added to supply extra data analysis information. Figure S-1 shows that the product temperature plateau due to mannitol crystallization during the freezing step can be better monitored using the wireless temperature sensors when higher concentrations are freeze-dried. Figure S-2 shows the peak intensity of the Raman ice band (215 cm-1) versus process time during experiment 1. Figure S-3a,b shows the NIR data analysis during the freezing step of experiment 1. Figure S-4a,b shows the Raman and NIR spectral changes during secondary drying. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review May 14, 2009. Accepted July 17, 2009. AC9010414


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Importance of Using Complementary Process Analyzers for the

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