Characterizing an Amorphous System Exhibiting Trace Crystallinity: A

Dec 14, 2007 - Solid State Research Group, School of Pharmacy, and Department of Chemistry, University of Otago, New Zealand, Drug Discovery and Devel...
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Part of the Special Issue: Facets of Polymorphism in Crystals

Characterizing an Amorphous System Exhibiting Trace Crystallinity: A Case Study with Saquinavir

CRYSTAL GROWTH & DESIGN 2008 VOL. 8, NO. 1 119–127

Andrea Heinz,*,†,‡,§ Clare J. Strachan,‡ Faraj Atassi,4 Keith C. Gordon,⊥ and Thomas Rades† Solid State Research Group, School of Pharmacy, and Department of Chemistry, UniVersity of Otago, New Zealand, Drug DiscoVery and DeVelopment Technology Center (DDTC) and DiVision of Pharmaceutical Technology, Faculty of Pharmacy, UniVersity of Helsinki, Finland, and Eli Lilly and Company, Indianapolis, Indiana ReceiVed September 20, 2007; ReVised Manuscript ReceiVed NoVember 27, 2007

ABSTRACT: In this study, the ability of different analytical techniques to detect and characterize trace crystallinity in amorphous saquinavir was compared and the same techniques were used to investigate differences in the amorphous material before and after removal of the trace crystallinity by milling or heating. Saquinavir samples were analyzed for trace crystallinity and differences in the amorphous state by X-ray powder diffraction in combination with pair distribution function transforms, differential scanning calorimetry, modulated temperature differential scanning calorimetry, polarizing light microscopy, scanning electron microscopy, as well as Raman, near-infrared, and mid-infrared spectroscopy combined with multivariate analysis. X-ray powder diffraction and polarizing light microscopy were best suited to detecting small amounts of residual crystallinity and showed that these can easily be removed by heating or milling. Thermal analysis confirmed structural differences between amorphous saquinavir containing trace crystallinity and the milled and heated samples. Pair distribution function transforms of the X-ray powder diffraction data and the spectroscopic techniques in combination with multivariate analysis revealed differences in short-range and long-range order of the different samples. Raman spectroscopy was the most sensitive spectroscopic technique to detect structural changes induced by milling and heating saquinavir. Overall, the results suggest that the amorphous forms differ in their degree of disorder and molecular bonding arrangements. The study shows that significant insight into trace crystallinity and short-range order in amorphous material can be obtained by using a variety of physical characterization methods and data analysis techniques. Introduction Molecular solids can be classified in terms of structure on the basis of the extent of order in the molecular networks. In crystalline materials, structural units (unit cells) that are repeated in a regular manner form a well-defined lattice. Unlike crystalline compounds, which exhibit orientational and positional longrange order in all three dimensions of space, amorphous materials show no long-range order of molecular packing. However, local molecular assemblies characterized by shortrange order may occur in the amorphous form.1,2 On the supramolecular level further variation among the crystalline and amorphous forms of a solid compound is possible. Due to differences in molecular interactions crystalline materials may exist in different polymorphic or solvate forms. The term polymorphism describes the ability of the crystalline material to form different lattice structures and/or different molecular conformations without undergoing changes in its chemical composition. When solvent molecules are incorporated into the crystal lattice, the resulting structures are designated solvates.1,3 The term polyamorphism describes the existence of at least two distinct amorphous forms of the same solid compound, * Corresponding author. Tel: 64 3 479 5285. Fax: 64 3 479 7034. E-mail: [email protected]. † School of Pharmacy, University of Otago. ‡ Drug Discovery and Development Technology Center (DDTC), University of Helsinki. § Division of Pharmaceutical Technology, University of Helsinki. 4 Eli Lilly and Company. ⊥ Department of Chemistry, University of Otago.

which represent discrete phases from a thermodynamic perspective and are thus separated by a clear phase transition.4 Firstorder amorphous-to-amorphous phase transitions have been observed for inorganic materials such as ice, SiO2, and Ge upon compression of the compound as a response to the densification,5–8 but in the pharmaceutical field, the occurrence of true polyamorphism according to the abovementioned definition has not yet been reported. However, several studies, e.g., on indomethacin, piroxicam, and cefditoren pivoxil, have revealed the existence of different amorphous forms of the pharmaceutical solid depending on the preparation technique.9–13 These amorphous phases prepared by quench-melting, milling/grinding, or spraydrying exhibit differences in their physical and chemical properties and, hence, in their pharmaceutical performance. They are sometimes referred to as pseudopolyamorphs, even though this term is not generally accepted, and they represent kinetic states varying primarily in their energetic difference to the equilibrium supercooled liquid condition. Typically, pharmaceuticals are manufactured in a stable crystalline form because the amorphous form tends to convert to the crystalline form because of its thermodynamic instability. However, despite this clear disadvantage, the amorphous state has attracted considerable interest in recent years. A higher molecular mobility of an amorphous substance as compared to its corresponding crystalline counterparts, for instance, may improve the solubility and dissolution rate of poorly soluble crystalline drug candidates and thus may result in enhanced transport characteristics and gastrointestinal absorption.14–16

10.1021/cg700912q CCC: $40.75  2008 American Chemical Society Published on Web 12/14/2007

120 Crystal Growth & Design, Vol. 8, No. 1, 2008

Whichever solid-state form is chosen, it is important for several reasons, including stability and patenting issues, to be able to ensure that this form is not contaminated by other solid-state forms.17 In this context, it is also crucial to determine the effects of processing on the pharmaceutical compound as different preparation and manufacturing techniques may induce solidstate conversions (e.g., crystalline to amorphous, polymorph transformations) or in case of amorphous materials smaller changes in the molecular structure and physicochemical properties of the drug.10 Thus, solid-state changes must be closely monitored and trace levels of contaminating solid-state forms detected. A variety of analytical techniques is available for such characterization. The particulate level is probed using X-ray powder diffraction (XRPD), thermal methods, and microscopic techniques. XRPD is considered the gold standard in the detection of differences in molecular order, e.g., periodicities of atoms or molecules in crystals, and allows the distinction between crystalline polymorphs and amorphous substances. Recently, pair distribution function (PDF) transforms of experimental XRPD data have been used for the identification of characteristic atom-to-atom distances within pharmaceutical materials that lack long-range order, or where short-range order is not reflected in the longrange order of the crystal (amorphous, disordered crystalline, and partially crystallized materials). It has been reported that PDF analysis allows a better understanding of structural differences in amorphous samples that have been prepared by different methods.11,12,18,19 Differential scanning calorimetry (DSC) and in particular modulated temperature differential scanning calorimetry (MTDSC) provide comprehensive insights into the glass-transitional behavior of amorphous substances and polymorphic properties of crystalline compounds, such as recrystallization and melting temperatures. MTDSC shows a high sensitivity and resolution and enables the deconvolution of complex and overlapping thermal events. It has previously been used to detect small amounts of amorphous material in crystalline systems and to describe differences in amorphous forms created by different preparation techniques.5,20–22 With the help of thermogravimetric analysis (TGA), information about moisture content and degradation temperatures of different solid-state forms can be obtained. Polarizing light microscopy (PLM) can be utilized to distinguish between isotropic amorphous materials and anisotropic crystalline substances because of the effects of birefringence that occur in crystalline (with the exception of the cubic crystalline state) but not in amorphous systems. Even small amounts of crystallinity can be detected, and solid-state transitions associated with changes in birefringence can be observed when PLM is coupled to a hot stage. Scanning electron microscopy (SEM) is employed to investigate the morphology of different solid-state forms of a solid compound. Techniques that are used to examine the properties of the drug on the molecular level include vibrational spectroscopy, such as attenuated total reflectance (ATR), diffuse reflectance infrared Fourier transform (DRIFTS), near-infrared (NIR), and Raman spectroscopy, as well as solid-state nuclear magnetic resonance (SSNMR) spectroscopy.1,23 In recent years, spectroscopic techniques have been extensively used to investigate polymorphism and amorphous materials.24–27 However, the spectral differences between different solid-state forms may be subtle. To overcome this problem, multivariate methods such as principal component analysis (PCA) have been used to a greater extent to extract qualitative information from the spectra,

Heinz et al.

Figure 1. Structural formula of saquinavir.

monitor solid-state changes, and visualize molecular-level differences between different samples.10,28 The pharmaceutical model compound that was chosen for this study is saquinavir, a protease inhibitor that prevents the proliferation of the human immunodeficiency virus (HIV). The drug exhibits extended (pseudo)polymorphism, and more than 15 solid-state structures have been identified and described by the manufacturer. Amorphous saquinavir is prepared from a crystalline ethyl ester solvate, termed form F, which contains the solvent in open, tunnel-shaped cavities. Upon rapidly evaporating the solvent, the crystal lattice collapses, leading to a disordered structure of the drug substance. In contrast to many other pharmaceutical compounds that are highly unstable in the amorphous form, the free base of saquinavir remains amorphous even at stress stability conditions. However, during incorrect preparation of amorphous saquinavir from form F, small amounts of trace crystallinity may remain. Thus, the aim of the study was to evaluate the ability of different analytical techniques to detect and characterize trace crystallinity in saquinavir. In addition, the analytical techniques in combination with various data analysis tools were used to compare the amorphous material before and after treatment by milling or heating to obtain information about potential differences in the amorphous state. Experimental Section Materials. Saquinavir (Figure 1) was kindly supplied by F. Hoffmann-La Roche Ltd. (Basel, Switzerland) and analyzed as received. Although the marketed product contains amorphous drug substance, the predominantly amorphous research sample characterized in this study showed crystalline impurities originating from the desolvate of an ethyl ester solvate used for the production of amorphous saquinavir. In the following, this sample is referred to as “supplied research sample”, SRS. To investigate the effects of different preparation techniques on the solid state of saquinavir, we milled or heated the drug. Milling was performed for 5 min utilizing a planetary mill (Pulverisette 6, Fritsch GmbH, Idar-Oberstein, Germany). The rotation speed was 400 rpm and the ball to mass ratio was 10:1. Heating of the received material was carried out on an aluminum pan in a moisture analyzer (MA 100, Sartorius AG, Göttingen, Germany) at 155 °C for 3 min. The heated material was slowly cooled to ambient temperature in a desiccator over phosphorus pentoxide to prevent atmospheric moisture condensation and then lightly ground with a mortar and pestle. All samples were analyzed on the day of preparation. Analytical Techniques. X-ray Powder Diffraction. Diffraction patterns were recorded using a θ-θ X-ray powder diffractometer (D8 Advance, Bruker AXS GmbH, Karlsruhe, Germany) equipped with Göbel mirror bent multilayer optics. The positions of the X-ray tube and detector were checked by using zero point definition of the 2θ scale. This was performed by using a slit in the center of the goniometer and a copper filter in front of the detector. A rocking curve was

Saquinavir: Amorphous System Exhibiting Crystallinity determined from -1.00° 2θ to 1.00° 2θ, and the angle of maximum intensity was measured. The measurements were performed in symmetrical reflection mode with Cu KR radiation (λ ) 1.54 Å) at 40 kV and 40 mA. The powder samples were scanned in the angular range of 3° 2θ to 60° 2θ with a step size of 0.02° 2θ and a count time of 2 s per step. In addition to conventional XRPD, variable-temperature XRPD was performed on saquinavir SRS employing an implemented heating system in the temperature range between 25 and 150 °C with a step size and count time as described above. The heating rate was 3 K min-1 and diffractograms were recorded between 3° 2θ and 35° 2θ every 5 °C after the samples had been kept isothermal at the temperature of interest for 5 min. Polarizing Light Microscopy. Polarizing light microscopy (DAS Mikroskop Leica DMLB, Leica Mikroskopie und Systeme GmbH, Wetzlar, Germany) was used to determine whether the samples contained birefringence. They were examined using a 50-fold magnification. The polarizing light microscope was coupled to a Mettler Toledo FP900 thermo system (Mettler-Toledo AG, Greifensee, Switzerland) comprising a Mettler Toledo FP90 central processor and Mettler Toledo FP82-HT microscope hot stage to monitor solid-state changes in saquinavir SRS between 25 and 120 °C at a heating rate of 3 K min-1. Scanning Electron Microscopy. The samples were mounted on aluminum stubs with double-sided carbon tape and coated with a thin layer of gold-palladium in a BioRad E5100 SEM coating system sputter coater (BioRad Microscience Ltd., Hertfordshire, U.K.) prior to analysis. SEM was performed using a Cambridge Instruments S360 scanning electron microscope (Cambridge Instruments, Cambridge, U.K.) with a beam acceleration voltage of 8 kV. Digital images were captured with a Dindima Image Slave frame grabber (Dindima Group Pty Ltd., Ringwood, Vic, Australia). Karl Fischer Titrimetry. A Karl Fischer titrator (Mettler DL 35, Greifensee, Switzerland) was used to determine the water content (expressed as w/w %) of three independent samples (n ) 3) of each set of samples (saquinavir SRS, milled, heated) directly after preparation. The standard sample for calibration was analytical grade sodium tartrate hydrate (water content 15.7%). Thermal Analysis. DSC and MTDSC experiments were performed on a Mettler DSC823e (Mettler-Toledo AG, Greifensee, Switzerland). Nitrogen was used as purge gas at a flow rate of 50 mL min-1. The samples were measured in crimped aluminum pans. The instrument was calibrated for temperature using indium. Standard DSC experiments were carried out using the Mettler DSC823e in its conventional mode at a heating rate of 10 K min-1 with the temperature ranging from 25 and 180 °C. Each sample was measured in triplicate. High-speed DSC measurements were performed in conventional mode between 25 and 350 °C at a nominal heating rate of 300 K min-1 to investigate a possible melting of residual crystalline material in the samples. For MTDSC measurements the instrument was operated at TOPEM mode (MTDSC mode of Mettler-Toledo) between 80 and 130 °C. The following parameters were chosen: a modulation amplitude of ( 0.159 K and a modulation period randomly varying between 15 and 30 s with an underlying heating rate of 2 K min-1.29,30 Glass transition temperatures (Tg) and changes in heat capacities (∆Cp) at the Tg were determined from the MTDSC data using the STARe software (MettlerToledo AG, Greifensee, Switzerland). The glass transition was determined as midpoint of the step change in the heat capacity. A one-way ANOVA (Microsoft Excel 2002, v. 10) was carried out to evaluate the differences in Tg and ∆Cp between the three sets of saquinavir samples (saquinavir SRS, milled, heated). Each set of samples consisted of five individual samples that were measured once. Degradation of saquinavir SRS was investigated by TGA using a TA-TGA Q50 (TA Instruments-Waters LLC, New Castle, DE, USA) with nitrogen as a purge gas at a flow rate of 60 mL min-1. The samples were heated in open aluminum pans (TA Instruments) from 25 to 480 °C at a heating rate of 10 K min-1. Data were analyzed using TAUniversal Analysis 2000 software (TA Instruments). Raman Spectroscopy. Raman spectra were recorded on a Bruker RFS 100/S FT-Raman spectrometer (Bruker Optik, Ettlingen, Germany) using a diode pumped Nd:YAG laser with an excitation wavelength of 1064 nm and a laser power of 500 mW. The spectra were acquired in the spectral range between 10 and 3500 cm-1 and each spectrum was the average of 200 scans. Six independent samples of each set of samples (saquinavir SRS, milled, heated) were analyzed. The interferograms were apodized with the Blackman-Harris 4-term function

Crystal Growth & Design, Vol. 8, No. 1, 2008 121 and subjected to Fourier transformation, yielding spectra with a resolution of 4 cm-1. Near-Infrared Spectroscopy. NIR spectra were recorded using a Vector 22/N FT-NIR spectrometer (Bruker Optik, Ettlingen, Germany) with a tungsten light source and a PbS detector. The samples were placed in glass vials and measured in reflectance mode over a wavelength range from 833 to 2857 nm (12 000 to 3500 cm-1). For each spectrum 128 scans were averaged. Six independent samples of each set of samples (saquinavir SRS, milled, heated) were measured. The interferograms were apodized with the Blackman-Harris 3-term function and subjected to Fourier transformation, yielding spectra with a resolution of 4 cm-1. Infrared Spectroscopy. Infrared spectra were recorded on a Bruker IFS 28 Fourier transform infrared spectrometer (Bruker Optik, Ettlingen, Germany) employing an ATR accessory with a ZnSe crystal (Pike Technologies, Madison, WI, USA), a KBr beam splitter, a globar source, and a DTGS detector. The samples were measured over a spectral range from 680 cm-1 to 4000 cm-1 and for each spectrum 32 scans were averaged. Six independent samples of each set of samples (saquinavir SRS, milled, heated) were analyzed. The interferograms were apodized with the Blackman-Harris 3-term function and subjected to Fourier transformation yielding spectra with a resolution of 2 cm-1. The ATR spectra were converted to absorbance spectra using OPUS software (v. 5.0, Bruker Optik, Ettlingen, Germany). Pair Distribution Function Analysis. PDF analysis is used to analyze the local structure of crystalline, quasicrystalline, or amorphous materials on the basis of their total scattering patterns. The PDF gives peaks corresponding to the distribution of atom-to-atom distances and, therefore, it reflects the structure of the investigated material. The PDF is obtained via Fourier transformation of normalized scattered X-ray or neutron intensities (eq 1), where the scattering data contain both Bragg and diffuse scattering contributions, and can be defined directly in real-space in terms of atomic coordinates.19 G(r) ) 4π[F(r) - F0] )

2 π

∞

∫ Q[S(Q) - 1]sin(Qr)dQ

(1)

0

The function G(r) is the atomic PDF, S(Q) is the total scattering structure function and is determined from the measured diffraction intensity. The study of local structure can be directly done by studying S(Q) in the reciprocal space or by Fourier transforming S(Q) to real space and studying the PDF. F(r) is the microscopic pair density, F0 is the average number density, and Q is the magnitude of the scattering vector.31 In this study, the experimental XRPD data were corrected using standard methods19,31 to obtain the total scattering structure function, S(Q), and the PDF, G(r). This was done using the PDFgetX2 software package.32 Multivariate Data Analysis. PCA was used to extract information from the experimental data obtained by vibrational spectroscopy and visualize possible differences in the amorphous saquinavir samples. All calculations were performed using Simca-P multivariate analysis software (v. 10.5, Umetrics AB, Umeå, Sweden). Prior to PCA, the spectral data were subjected to different established pre-treatment methods and different spectral regions were tested. For Raman spectroscopy, the spectral range between 1400 and 1725 cm-1 was chosen and the spectra were standard normal variate (SNV) corrected and mean centered. The spectral region between 1600 and 2500 nm of the NIR spectra was analyzed after SNV correction and mean centering of the data. The area between 1800 and 1960 nm containing a broad band with a maximum at 1940 nm that is due to water was excluded from the PCA model to minimize the effects of absorbed moisture. In the case of IR spectroscopy, the area between 1470 and 1710 cm-1 was chosen for multivariate analysis and the spectra were pre-processed using SNV correction and mean centering.

Results and Discussion X-ray Powder Diffraction. The X-ray diffractograms of all three sets of samples (saquinavir SRS, milled, heated) showed a halo pattern characteristic of amorphous materials (Figure 2). However, although the heated and milled samples were completely X-ray amorphous, the distinct peak at 4.5° 2θ occurring

122 Crystal Growth & Design, Vol. 8, No. 1, 2008

Figure 2. Experimental XRPD patterns of (a) crystalline saquinavir form F, (b) saquinavir SRS, (c) saquinavir that was milled for 5 min, and (d) heated saquinavir. Diffractograms are offset for clarity.

in the diffractograms of saquinavir SRS indicated the presence of residual crystallinity. As can furthermore be seen in Figure 2, the crystalline ethyl ester solvate, form F, which is the starting material for the preparation of amorphous saquinavir, exhibits a similar peak at 4.25° 2θ in the X-ray pattern (XRPD data of form F was kindly provided by the manufacturer). It has been observed before that during preparation of amorphous saquinavir by evaporation of ethyl ester from form F a peak shift from 4.25° 2θ to 4.5° 2θ takes place before the material turns amorphous.33 When a solvent is lost from a crystalline solvate structure, in some cases, the three-dimensional order of the crystal lattice may be retained, resulting in an isomorphous desolvated form that represents a high-energy state relative to the original solvate structure.34 According to this theory, it is therefore likely that upon evaporation of ethyl ester from form F, a small proportion of the channel structure remains intact and an isomorphic desolvate is formed with structural relaxation reflected in a peak shift to 4.5° 2θ. Thus, the residual crystallinity found in saquinavir SRS may be attributed to an isomorphic desolvate of the ethyl ester solvate form F. Isomorphic desolvates and similar structural relaxation have been observed for other solvates exhibiting channel structures.34,35 The diffraction patterns show that the peak at 4.5° 2θ, and thus residual crystallinity, can be easily removed by milling saquinavir for 5 min or heating the material to 155 °C. Therefore, it can be assumed that irreversible destruction of the residual crystal lattice is achieved by either introducing mechanical stress through milling or increasing the mobility of the saquinavir molecules by heating. In addition to conventional XRPD, variable-temperature XRPD experiments of saquinavir SRS revealed that the peak at 4.5° 2θ disappears at temperatures between 100 and 110 °C (data not shown), indicating that as soon as the predominantly amorphous material changes from the glassy to the less viscous rubbery state at the Tg (about 104 °C, see below) the molecular mobility increases and the residual crystalline structure collapses. PDF transforms are presented as weighted probability of finding an atom separated by a distance r from a reference atom, and PDF peaks thus represent commonly occurring interatomic distances.18 In this study, PDF analysis was used to investigate structural changes in saquinavir SRS upon milling and heating, as well as structural differences between the crystalline form F and the three predominantly amorphous and amorphous forms

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Figure 3. Pair distribution function transforms of the XRPD data of crystalline saquinavir form F, saquinavir samples SRS from the manufacturer, milled saquinavir, and heated saquinavir showing molecular packing distances up to 30 Å.

based on the distribution of atom to atom distances within the different solid-state forms. Figure 3 shows the first PDF peaks corresponding to the nearest neighbor (NN), the next nearest neighbor (NNN), and further atomic interactions up to 30 Å.18 Overall, comparing the crystalline form F with the three amorphous forms, it becomes obvious that NNN interactions greater than approximately 15 Å occur to a considerably lesser extent in the amorphous samples. The loss of long-range, intermediate-range, and to some extent short-range order compared to the parent crystalline form is characteristic of amorphous materials.1,2,18 Comparing the crystalline form F with the predominantly amorphous saquinavir SRS a similar molecular packing motif for NN interactions up to 12 Å can be found in both forms indicating that both exhibit a similar short-range order. Considering saquinavir SRS was prepared from the crystalline form F by evaporation of the solvent ethyl ester it is likely that the loss of solvent, which is accompanied by a collapse of former solvent channels, is causing a continuous loss of long-range order. An amorphization process characterized by a continuous loss of long-range order while some short-range order of the parent crystalline form is retained has been described before and is referred to as kinetic disordering.18 While interactions below 12 Å are similar to those in form F, interactions greater than 12 Å in saquinavir SRS much more resemble those in the milled and heated amorphous forms. As mentioned before, this may be attributed to the loss of higher-range order of the parent crystalline form upon formation of the amorphous form. As saquinavir SRS is milled or heated the local NN configuration changes to a new amorphous packing, which also differs from that of the crystalline form F. The three NN peaks that correspond to approximate atom-to-atom distances of 2.1, 5.1, and 7.0 Å are shifted to 2.5, 5.2, and 7.3 Å, respectively, upon milling and 2.7, 5.3, and 7.4 Å, respectively, upon heating. These results indicate that the degree of disorder of the amorphous material increases when saquinavir SRS is subjected to heating and milling. However, further studies are suggested to explore the long-range distance differences between the saquinavir samples using PDF analysis on XRPD data obtained with a higher-resolution X-ray source. Polarizing Light Microscopy. When analyzed by PLM the saquinavir received from the manufacturer showed small areas of birefringence, indicating residual crystallinity. Consistent with the XRPD results the milled and heated samples were com-

Saquinavir: Amorphous System Exhibiting Crystallinity

Crystal Growth & Design, Vol. 8, No. 1, 2008 123 Table 1. Thermodynamic Properties of Different Saquinavir Samples Obtained by MTDSC (n ) 5)

saquinavir SRS saquinavir milled saquinavir heated

Tg (°C)

(S.D. (°C)

∆Cp (J g-1 K-1)

(S.D. (J g-1 K-1)

104.6a 103.8 103.6

0.2 0.2 0.4

0.43a 0.48 0.51

0.02 0.02 0.04

a Significantly different from saquinavir milled and heated (p < 0.05).

Figure 4. SEM images of (a) saquinavir SRS, (b) milled saquinavir, and (c) heated saquinavir.

pletely amorphous and no birefringence was observed. Hot stage polarizing light microscopy revealed that the birefringence starts to disappear at the Tg of saquinavir, which is in agreement with the results obtained by variable-temperature XRPD. Scanning Electron Microscopy. SEM was used to obtain information about the morphology of saquinavir before and after removal of trace crystallinity by melting and heating (Figure 4). Saquinavir as received consisted of agglomerates of fused rod-like particles with sizes up to 50 µm and smaller micro-

particles of approximately 1-2 µm (Figure 4a). Upon milling, the morphology of saquinavir changed to smaller particles with sizes up to 10 µm, which were irregular in shape (Figure 4b). Some of the rod-like structures still existed; however, they were reduced in size and no big agglomerates were observed. Upon heating saquinavir to 155 °C and subsequent cooling to ambient temperature, a brittle glass was formed. After gently grinding the material, we found particles of different sizes up to 400 µm with a smooth surface (Figure 4c). Karl Fischer Titrimetry. The water content of the saquinavir samples was determined to be 1.7% ( 0.1% for saquinavir SRS, 1.4% ( 0.1% for milled saquinavir, and 0.5% ( 0.2% for heated saquinavir. Apparently, upon milling and heating, some of the water initially present in the samples is evaporating. During milling, temperatures of between 32 to 36 °C were measured in the milling jars using an IR thermometer (KM814, Comark Limited Inc., Stevenage, Hertfortshire, U.K.). Thermal Analysis. Amorphous saquinavir has previously been studied by MTDSC and the focus was placed on the determination of the Tg and characterization of the effects of moisture uptake on the Tg.29,30 In this work, however, the emphasis was put on the comparison of the thermal behavior of differently treated saquinavir samples (saquinavir SRS, milled, heated) to allow conclusions about structural differences between the samples. For this purpose, the Tg and the ∆Cp values at the Tg were determined. The Tg is an important measure for chemical and physical stability of an amorphous material and changes in heat capacity at the Tg may provide information about residual crystallinity.14 Conventional DSC in the temperature range between 25 and 180 °C was used to estimate the Tg (data not shown). The thermograms of all samples contained an endotherm between 50 and 90 °C resulting from water loss. In addition, a smaller endotherm with a maximum at about 104 °C and a discontinuity in the baseline before and after the thermal event were observed. Both are indicative of a glass transition.22 However, from the conventional DSC traces, it is difficult to determine the exact temperature range over which the glass transition is occurring as the glass transition is accompanied by an endothermic relaxation. Thus, MTDSC experiments were carried out to separate the reversing glass transition and the non-reversing relaxation endotherm. The MTDSC response of a saquinavir sample prepared by milling for 5 min shows the total, reversing, and non-reversing heat flow (Figure 4). A glass transition at 103.8 °C can be found in the reversing trace, and the nonreversing signal shows a relaxation endotherm with an onset at 100.1 °C. The reversing trace of each sample was used to determine the Tg and the ∆Cp at the Tg. The results are summarized in Table 1. Comparing the three sets of samples by one-way ANOVA revealed statistically significant differences between the thermal properties of saquinavir SRS and those of the other two sets of samples. Saquinavir SRS showed significantly higher Tg (104.6 °C ( 0.2 °C) and significantly lower ∆Cp values (0.43 J g-1 K-1 ( 0.02 J g-1 K-1). The smaller ∆Cp values as

124 Crystal Growth & Design, Vol. 8, No. 1, 2008

Figure 5. MTDSC response (total, reversing, nonreversing heat flow) of a saquinavir sample prepared by milling.

compared to the milled and heated samples support the XRPD findings that trace crystallinity was present. As previously mentioned, this trace crystallinity can be removed by milling and heating the material, which leads to an increase in the ∆Cp at the Tg. The thermal properties of the milled and the heated samples appear to be similar. The differences in the Tg values between the three sets of samples can be assumed to be due to structural differences in the amorphous state (e.g., differences in the degree of disorder and molecular interactions such as hydrogen bonding) rather than effects of water in the samples influencing the Tg. Even though it is well-known that water has an influence on the Tg, plasticizing effects that may lower the Tg occur only under certain conditions: in hermetically sealed pans from which water cannot escape and when the Tg is below the temperature needed to remove the water.15,30 However, in the case of saquinavir, the Tg value was obtained in crimped pans and the water had already evaporated when the glass transition occurred. This is supported by the endotherm of water loss that can be observed between 50 and 90 °C as has been described previously by Royall et al.30 High-speed DSC was performed to detect residual crystallinity. Because of the high heating rates, recrystallization events induced by heating are usually overrun. Thus, any melting event during the experiments can be attributed to residual crystallinity in the sample. No melting endotherm was observed, which is in good agreement with the variable-temperature XRPD and hot stage PLM results obtained for saquinavir SRS where residual crystallinity was already removed at the Tg of about 104 °C. However, a large endotherm was observed between 200 and 350 °C, indicating degradation of saquinavir, which was also supported by the TGA results. To ensure heating saquinavir to 155 °C is not causing any degradation. A strong weight loss of the material indicating degradation was observed between 200 and 460 °C (data not shown). Thus, it is reasonable to assume that differences in the milled and heated amorphous saquinavir samples are not caused by degradation. The TGA scans furthermore revealed that the water content of saquinavir SRS was 1.7%, which is consistent with the results obtained by Karl Fischer titrimetry. Vibrational Spectroscopy. Raman Spectroscopy. The Raman spectra of saquinavir SRS, the milled, and the heated samples were very similar, with only one spectral difference visible (Figure 5). A merged double band that can be assigned to CdO stretching vibrations shows its highest intensity at 1662

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Figure 6. Raman spectra of (a) saquinavir SRS, (b) saquinavir milled for 5 min, and (c) heated saquinavir in the spectral range between 1100 and 1725 cm-1. The spectra are offset for clarity.

cm-1 in saquinavir SRS. The intensity decreases at 1662 cm-1 in the milled and heated samples such that the maximum of the merged band is shifted to 1670 cm-1 in the heated samples. In agreement with the XRPD and MTDSC results, the decreased intensity and shift of the merged band indicate structural changes in saquinavir SRS induced by milling and heating. To check that the spectral differences in the CdO stretching region were not related to the differences in the water content of the samples but were actually due to structural differences in the amorphous phases. In the first experiment, Raman spectra of saquinavir SRS that had been stored over P2O5 for three days and whose water content was determined to be 0.5% ( 0.1% were recorded. In the second experiment, a paste of saquinavir and distilled water was prepared and analyzed by Raman spectroscopy. It could be confirmed that the spectra of both the saquinavir SRS stored over drying agent and the spectra of the paste of saquinavir SRS with distilled water showed the same spectral features in the region between 1640 and 1700 cm-1. It can thus be concluded that the spectral differences of saquinavir SRS compared to the heated and milled samples are not caused by differences in the water content of the three sets of samples. The PCA analysis of the Raman spectra confirmed that there are differences between the milled samples, the heated samples, and saquinavir SRS. Two principal components (PCs) explained 86.4% of the spectral variation. The spectral differences in the CdO stretching region at 1660 cm-1 are described by the first PC (Figure 7a). The scores plot in Figure 7b shows separate clusters for the different sets of samples (saquinavir SRS, milled, heated). The separation of the samples is accomplished through the first PC. This suggests that there are continuous differences in the degree of disorder and not distinct intermolecular bonding and/or conformation differences per se for all three sets of samples. In the light of the SEM findings and consistent with the results obtained by PDF analysis of the XRPD data, the results of Raman spectroscopy combined with multivariate analysis indicate that the degree of disorder increases upon milling or heating saquinavir SRS. According to the scores plot, heating leads to the most disordered material and the heated samples thus cluster the farthest apart from saquinavir SRS. Because changes in the CdO stretching region between the Raman spectra of the three sets of samples can be observed, differences on the molecular level are likely to involve changes in the

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Figure 7. (A) Loadings and (B) scores plots generated from the Raman spectra of saquinavir SRS, milled saquinavir, and heated saquinavir by PCA. Loadings are offset for clarity.

hydrogen bonding between saquinavir molecules associated with CdO groups. When the near order in the samples changes and the degree of disorder increases upon milling and heating these hydrogen bonds might be disrupted. NIR Spectroscopy. No major spectral differences between the NIR spectra of the three sets of samples (saquinavir SRS, milled, heated) were observed in the spectral range from 1100 to 2600 nm (data not shown). As NIR spectroscopy is highly sensitive to the presence of water and hydrogen bonding between water and components of the sample, characteristic water bands with maxima at 1190, 1450, and 1940 nm were found.36 For PCA, the spectral region between 1600 and 2425 nm was investigated and water bands were excluded from the model. With two PCs, 90.7% of the spectral variation could be explained. According to the first PC versus the second PC scores plot, the first PC distinguished the heated material from the other two sets of samples. However, even though the second PC slightly contributed to the separation between milled and saquinavir SRS, no clear clustering could be observed.

IR Spectroscopy. No major differences between saquinavir SRS, heated saquinavir, and milled saquinavir were visible in the IR spectra. PCA was performed in the region from 1470 to 1710 cm-1, and with two PCs, 92.6% of the spectral variation could be explained. The PCs revealed differences between the spectra in the CdO stretching region around 1660 cm-1. However, no clustering of the different sets of samples could be observed in the scores plot (data not shown). Thus, with a combination of IR spectroscopy and PCA, it was not possible to distinguish between the different sets of samples and visualize molecular-level differences. Discussion The various analytical techniques that have been used to probe the particulate and molecular levels of differently prepared predominantly amorphous and amorphous forms of saquinavir showed different sensitivities toward differences in the molecular properties of the samples. In recent years, XRPD has extensively

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been used as a standard technique in solid-state characterization to distinguish between crystalline and amorphous forms and to identify different crystalline polymorphs of pharmaceutical compounds. X-ray amorphous systems exhibit a characteristic halo pattern in the diffractogram, whereas crystalline materials result in distinct diffraction peaks, which renders it possible to easily identify residual crystallinity. In this study, small amounts of crystallinity present in saquinavir SRS could be detected using XRPD and PLM. They are most likely attributable to the isomorphic desolvate of an ethyl ester solvate, form F, from which the amorphous saquinavir was produced. Coupled to hot stages both methods revealed that the residual crystallinity can be removed by heating the predominantly amorphous material to its Tg of about 104 °C. The increase in molecular mobility upon transition of amorphous saquinavir from the glassy to the rubbery state, most likely leads to a collapse of residual amounts of crystal lattice and eventually to the formation of a disordered amorphous structure. Preparation techniques that are widely used to create amorphous materials include milling and melting.10,14,37 XRPD and PLM confirmed that the trace crystallinity can indeed be removed by milling or heating saquinavir, which is equivalent to melting a crystalline material. Both preparation methods resulted in X-ray amorphous materials showing no birefringence in PLM. SEM revealed that saquinavir SRS consisted of agglomerates of fused rod-like particles. These are likely to be the remains from the preparation of the material by evaporating the solvent ethyl ester from the crystalline solvate form F where the inner channel structure of the particles collapsed, whereas their outer shape most likely only slightly changed (according to the manufacturer form F exhibits elongated needles).33 Because of the destruction of the channel structure, the material became amorphous and was thus nonbirefringent in PLM. Birefringence was observed in only very few particles where the channel structure had remained intact. During milling, the agglomerates of saquinavir SRS were broken into smaller particles of different shapes. No birefringence was observed, indicating that the milling process had destroyed the residual channel structures and thus had removed residual crystallinity. However, as the particles get smaller upon milling, it also has to be taken into account that the path length of light through a particle decreases, which may make it more difficult to observe residual birefringence. In contrast to the milled particles, which possessed an uneven surface, the amorphous saquinavir prepared by heating showed a smooth surface due to the heating process above the Tg of saquinavir SRS, which caused the drug particles to become less viscous and lose their original shapes to eventually form a brittle glass. Residual crystallinity was removed during heating saquinavir SRS, and the heated material showed no birefringence in PLM. PDF analysis provided a more detailed insight into the structural changes in saquinavir SRS upon milling and heating on a molecular level. Furthermore, structural differences between the crystalline form F and the three forms analyzed in this study were found. Saquinavir SRS exhibits a short-range order similar to that of the crystalline form F, whereas the long-range order is comparable with that of the milled and heated amorphous forms. The loss of long-range order compared to the crystalline form is characteristic of amorphous materials.1,2,18 Considering saquinavir SRS is prepared from the crystalline form F by evaporation of the solvent ethyl ester it is likely that the loss of solvent, which is accompanied by a collapse of former solvent channels, is resulting in a continuous loss of long-range order. An amorphization process characterized by a continuous loss

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of long-range order while some short-range order of the parent crystalline form is retained is referred to as kinetic disordering.18 The X-ray powder diffractograms and the results of PLM showed that the amorphization process was not complete in the case of saquinavir SRS as trace crystallinity was observed. Upon milling and heating, changes in the short-range order compared to the crystalline form F and saquinavir SRS occur and indicate that the degree of disorder in the amorphous material increases because of mechanical stress or energy input. These findings are in agreement with previous studies that have revealed differences in the amorphous materials prepared by different methods.10,13,18,38 The results of the thermal analysis using DSC and MTDSC complement the findings of the PDF transforms of the XRPD data. Saquinavir SRS was found to be significantly different from the other two sets of samples in two thermal properties, the Tg and the ∆Cp at the Tg. It showed the highest Tg and the lowest ∆Cp at the Tg of all sets of samples. The lower Tg and higher ∆Cp values of the heated and milled forms could be explained considering both forms showed a higher degree of disorder according to the PDF analysis. A higher degree of disorder is likely to slightly lower the Tg as it increases molecular mobility. A statistically significant distinction between the heated and milled samples in terms of their thermal properties, however, was not possible even though the overall lowest Tg and highest ∆Cp values that were calculated for the heated form, indicating the highest degree of disorder for this amorphous form. The smaller ∆Cp at the Tg of saquinavir SRS supported by the XRPD results suggests that residual crystallinity is present in the samples. Detection of trace crystallinity was not possible using the spectroscopic techniques as the Raman, NIR, and IR spectra of saquinavir SRS, milled saquinavir, and heated saquinavir were very similar. However, in the Raman spectra, the decreased and shifted maximum intensity of two merged bands in the CdO stretching region after milling and, even more so, heating saquinavir SRS indicated structural differences between the predominantly amorphous and amorphous samples. These may be associated with changes in the hydrogen bonding arrangement between different saquinavir molecules. Milling and heating may, for example, break hydrogen bonding between molecules and lead to a less ordered system. This assumption is supported by the results of the PDF analysis of the XRPD data, which revealed that the degree of disorder increased after milling and heating. Also, this result is consistent with those of the thermal analysis where the lower Tg of milled and heated saquinavir indicates a higher degree of disorder. Multivariate analysis of spectroscopic data can be used to visualize molecular-level differences between crystalline and differently prepared amorphous forms. A previous study, for instance, investigated milled, heated, and spray-dried amorphous forms, as well as two crystalline polymorphs of indomethacin using Raman, NIR, and IR spectroscopy combined with PCA.10 It was found that all spectroscopic techniques are able to distinguish between the different crystalline and amorphous forms, with Raman spectroscopy being the most sensitive technique towards structural differences in the amorphous samples. In agreement with this previous work, in the current study, Raman spectroscopy in combination with PCA proved to be the most suitable for characterizing subtle differences in the differently prepared predominantly amorphous and amorphous samples. A clear distinction between the three forms was possible on the basis of the PCA scores plot, which showed separate clusters for each set of samples with the milled form

Saquinavir: Amorphous System Exhibiting Crystallinity

clustering closer to saquinavir SRS than the heated form. Again, these results are consistent with those obtained by PDF analysis of the XRPD data and the thermal analysis and indicate that the structural changes compared to the starting material are slightly bigger in the case of the heated samples. On the basis of NIR spectroscopy combined with PCA, a distinction between saquinavir SRS containing trace crystallinity and the two amorphous forms prepared by milling and heating was possible. However, no clear separation between the milled and heated amorphous samples was achieved. IR spectroscopy combined with PCA failed to differentiate between the differently prepared amorphous samples, because spectral variation due to structural changes was less than spectral variation from other sources. Conclusion This study investigated the ability of a wide range of analytical techniques to detect trace crystallinity and characterize differences in the molecular properties of differently prepared amorphous forms of the pharmaceutical compound saquinavir. It could be shown that small amounts of trace crystallinity present in saquinavir SRS from the manufacturer can be detected using XRPD and PLM. As confirmed by XRPD and PLM, milling and heating are appropriate methods to remove the residual crystallinity. Further analytical techniques and analysis tools proved suitable to characterize molecular-level differences between the predominantly amorphous and amorphous samples. PDF transforms of the XRPD data, MTDSC, and Raman spectroscopy in combination with PCA revealed differences in molecular properties of the samples and indicate that the degree of disorder increases upon milling and even more upon heating saquinavir SRS. PDF analysis furthermore revealed that saquinavir SRS is close to the parent crystalline form F in its short-range order, whereas the milled and heated samples differ from the crystalline form in both short-range and long-range order. Raman spectroscopy suggests that structural changes during milling and heating involve changes in the hydrogen-bonding pattern. Overall, the study shows that it is beneficial to use a combination of different analytical techniques and data analysis tools to detect trace crystallinity and investigate differences in solid-state properties of differently prepared amorphous forms, because results are complementary and allow a more comprehensive description of the solid state. Acknowledgment. The University of Otago (University of Otago Postgraduate Scholarship) and the Finnish Centre for International Mobility (CIMO) are acknowledged for financial support (A.H.). G. Gross (F. Hoffmann-La Roche Ltd.) is acknowledged for supplying an experimental batch of partly crystalline saquinavir and O. Grassmann (F. Hoffmann-La Roche Ltd.) and M. Hennig (F. Hoffmann-La Roche Ltd.) are thanked for providing experimental X-ray powder diffraction data of the crystalline form F of saquinavir. H. Rudolf (Martin Luther University Halle-Wittenberg, Germany) is acknowledged for help with the Raman, NIR, and IR measurements. T. Walsh is thanked for his contributions in providing the TGA and SEM data and M. Savolainen and Ch. Schmelzer are acknowledged for valuable comments.

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