Robert T. Forbes

Moisture induced polymorphic transition of mannitol and its morphological transformation

The effects of moisture on the polymorphic transition of crystalline mannitol were investigated. Mannitol has three polymorphic forms, and was classified as alpha, beta, and delta form, respectively, by Walter-Lévy (C.R. Acad. Sc. Paris Ser. C (1968) 267, 1779). The water uptake of delta form crystalline was greater than that of the beta form when each crystalline form was stored at 97%RH (25 degrees C). The different powder X-ray diffraction patterns obtained before and after humidification confirmed that a moisture induced polymorphic transition from the delta to beta form had occurred. Morphological changes were also observed with an increase in the specific surface area of the delta sample from 0.4 to 2.3 m(2)/g being found on exposure to humidity. Thus it was suggested that the observed higher hygroscopicity of the newly formed beta form arose from the gradual increase in the surface area with the polymorphic transition from the delta to beta form. When considering the mechanism of this polymorphic transition, the results from molecular modelling, cross-polarisation/magic angle spinning (CP/MAS) solid-state NMR spectra and scanning electron-micrographs suggest that water molecules act as a molecular loosener to facilitate conversion from delta to the beta form as a result of multi-nucleation.

Supercritical fluid processing of proteins: I: Lysozyme precipitation from organic solution

The solution enhanced dispersion by supercritical fluid (SEDS) process was used to evaluate the effect of the processing variables on the biological and physicochemical characteristics of lysozyme protein particles produced from an organic solution of dimethylsulfoxide (DMSO) using an experimental design procedure. The processing variables were temperature, pressure, solution concentration and the flow-rates of supercritical carbon dioxide and a protein solution. Solutions of hen egg lysozyme (0.5-1%, w/v) in DMSO were dispersed using supercritical carbon dioxide as the antisolvent, and particles precipitated in a particle formation vessel. The morphology, particle size and size distribution and biological activity of the protein were determined. The precipitates were also examined with high sensitivity differential scanning calorimetry (HSDSC) and high-performance cation-exchange chromatography. The amount of residual DMSO was determined using headspace gas chromatography. Particle size measurements showed the precipitates to be agglomerates with primary particles of size 1-5 microm, containing <20 ppm of residual solvent. The activity of the precipitates varied between 44 and 100% depending on the experimental conditions. The similarity of HSDSC data for unprocessed and processed samples indicated that the SEDS process does not cause major denaturation of lysozyme when prepared from DMSO solutions. By optimising of working conditions, the SEDS process can produce micron-sized particles of lysozyme with minimal loss of biological activity.

The characterization and comparison of spray-dried mannitol samples

Background: Following the production of spray-dried mannitol powders, it is essential that the polymorphic content of each individual product is completely characterized. The implications of the polymorphic behavior of mannitol are immense. The appearance or disappearance of a crystalline form within a dosage form can have costly repercussions and lead to a dosage form being withdrawn. Method: In this study, commercially available and laboratory-produced spray-dried mannitol products were characterized to establish the polymorphic content of each. Their polymorphic behavior was also characterized after laboratory scale pharmaceutical processes. Thermal analysis employed differential scanning calorimetry, thermogravimetric analysis, and isothermal microcalorimetry. Structural analysis of the samples was obtained using X-ray powder diffraction and Fourier transform Raman spectroscopy. Results: Structural analysis revealed that α- and β- polymorphic forms were present in the commercial samples and some contained a mixture of polymorphs. Reprocessing employing spray drying indicated α- to β- polymorphic transitions occurred within some of the samples. Conclusion: It is essential that preformulation studies where spray-dried mannitol products are to be employed must take into account its polymorphic behavior upon supply, processing, and subsequent storage.

The improved compaction properties of mannitol after a moisture-induced polymorphic transition

We have previously shown that by exposing one form of mannitol to high relative humidity, a moisture-induced polymorphic transition of mannitol with a concurrent change in particle morphology occurs [Int. J. Pharm. 247 (2002) 69]. In this paper, we propose that if these changes occur during a wet-granulation procedure, it may be possible to make bring about an in situ size-reduction of mannitol with compaction property enhancement. Powder X-ray diffraction and scanning electron microscopy confirmed that a polymorphic transition (the delta form forming the beta form) had occurred on wet-granulation, and that a concomitant morphology change resulted in an agglomerate consisting of filament-like fine primary crystals (delta-granule). The aim of present study was to evaluate the compression properties of this agglomerate. The compact compressed with delta-granules possessed a tensile strength 1.5 times higher than other mannitol samples. Heckel analysis indicated that the mannitol compression process proceeded by deformation without fragmentation and was thus particle size dependent. The delta-granule showed enhanced plastic deformability, due to its unique particle structure. Because the intrinsic compression properties of the polymorphs were similar, the primary particle size and specific surface area of mannitol were indicated to be the major contributing factors for the improved compaction behaviour, rather than the polymorphic transition. When using the delta-granule as an excipient for a tablet formulation containing a high amount of phenylpropanolamine hydrochloride (PPA) as a poorly compactable model drug, excellent tablets could be prepared without capping, whereas conventional mannitol produced capped tablets.

A Central Composite Design to Investigate the Thermal Stabilization of Lysozyme

AbstractPurpose. The formulation and processing of protein drugs requires the stabilization of the native, biologically active structure. Our aim was to investigate the thermal stability of a model protein, lysozyme, in the presence of two model excipients, sucrose and hydroxypropyl-β-cyclodextrin (HP-β-CD). Methods. We used high sensitivity differential scanning calorimetry (HSDSC) in combination with a central composite design (CCD). As indicators of protein thermal stability, the measured responses were the unfolding transition temperature (Tm), the onset temperature of the denaturation (T0), and the extrapolated onset temperature (To,e). Results. A highly significant (F probability <0.001) statistical model resulted from analysis of the data. The largest effect was due to pH (over the range 3.2-7.2), and the pH value that maximized Tm was 4.8. Several minor but significant effects were detected that were useful for mechanistic understanding. In particular, the effects of protein concentration and cyclodextrin concentration on Tm and To,e were found to be pH-dependent. This was indicative of the partially hydrophilic nature of protein-protein interactions and protein-cyclodextrin interactions, respectively. Conclusions. Response surface methodology (RSM) proved efficient for the modeling and optimization of lysozyme thermal stability as well as for the physical understanding of the protein-sugar-cyclodextrin system in aqueous solution.

Dissolution kinetics and solubilities of p-aminosalicylic acid and its salts

Abstract A series of metal and amine salts of p-aminosalicylic acid (PAS) were synthesized and their water solubilities and intrinsic dissolution rates investigated. Whilst the increased solubility of PAS in the presence of its major breakdown product m-aminophenol (MAP) dictated that equilibrium solubilities were unable to be determined, apparent solubilities at 10°C after 1 h equilibration were obtained. The order of decreasing solubility and intrinsic dissolution rate for metallic salts of PAS was potassium > sodium > calcium (low hydrate) > calcium (trihydrate) = magnesium. When data for PAS and its ammonium and ethanolamine salts were included, a direct relationship between log solubility ( C s ) and log dissolution rate (IDR) was observed. Since the apparent solubility of the potassium salt was only qualitatively known, by applying this solubility-dissolution rate relationship its solubility was predicted to be 2.5 M/I −1 at 10°C using the regression line log IDR = 1.06log C s -0.166 ( r = 0.9931, n = 7). The relationship could not be used where a phase change at the solid-liquid interface occurred. Thus, the solubilities of the tosylate, mesylate and sulphate salts of PAS could not be estimated since these salts reverted during dissolution to form PAS.

Integrity of crystalline lysozyme exceeds that of a spray-dried form

The development of proteins as therapeutic agents is challenging partly due to their inherent instabilities. Consequently, crystallisation and spray drying techniques were assessed to determine their effects on protein integrity using lysozyme as a model protein. Unprocessed, crystallised and spray-dried lysozyme were characterised by: thermal analysis using hot stage microscopy (HSM), differential scanning calorimetry (DSC), high sensitivity differential scanning calorimetry (HSDSC) and thermogravimetry (TGA); and spectroscopic analysis employing Fourier transform Raman (FT-Raman). Moisture contents were determined by TGA and Karl Fisher titration (KFT). Enzymatic assay measured biological activity. HSM showed no changes in crystals until complete melting. TGA and KFT indicated that spray-dried lysozyme contained a lower moisture content than crystals, hence the higher apparent thermal stability was shown by DSC. HSDSC revealed that crystallisation and spray drying did not affect the denaturation temperature of lysozyme in solution when compared with unprocessed material. However, in the solid state, FT-Raman spectra showed perturbation of the conformational structure of spray-dried sample, whereas crystal conformation remained intact. Enzymatic assay revealed increased activity retention of crystals compared with spray-dried powder. Hence, crystals maintained the conformational integrity and activity of lysozyme in solution.

Crystallisation of amorphous mannitol is retarded using boric acid.

An approach to inhibit the crystallisation of amorphous mannitol was investigated. Boric acid was selected as a model additive for a fundamental study of its ability to retard crystallisation and to facilitate characterisation of the properties of the amorphous solid. At concentrations above 5% (w/w) of boric acid, the DSC scans indicated that a totally amorphous solid could be prepared by cooling the melted pre-mixture under ambient conditions. An increase in the glass transition temperature (T(g)) was observed with a corresponding increase in boric acid content, and their relationship was well fitted by the Gordon-Taylor equation. This result suggested that mannitol and boric acid mixed homogeneously. The crystallisation profiles of the resultant amorphous compositions were best described by the Avrami-Eroféev equation (n=1/3), which indicated that random nucleation and three-dimensional crystal growth was the best-fitting mechanism of this crystallisation. The activation energy of crystallisation decreased with increasing boric acid content, indicating that the temperature dependency for crystallisation decreased with increasing boric acid content. Furthermore, the rate of crystallisation at 30 degrees C for mannitol alone was 7000 times higher than that of mannitol containing 7.5% (w/w) of boric acid.

Supercritical fluid processing of proteins: lysozyme precipitation from aqueous solution.

Aqueous solutions of hen egg lysozyme (3% w/v) were dispersed and precipitated by a homogenous mixture of supercritical carbon dioxide–ethanol using the Solution Enhanced Dispersion by Supercritical fluid (SEDS) process. The effects of different working conditions, such as temperature, pressure and the flow rates of the solution and ethanol, on the particle‐formation process were studied. The morphology, particle size and size distribution and biological activity of the protein were determined. The precipitates were examined with high‐sensitivity differential scanning calorimetry (HSDSC) and high‐performance cation‐exchange chromatography. Particle size measurements showed the precipitates to be aggregates with primary particles of size 1–5 μm. The similarity of HSDSC data for unprocessed and processed samples indicated that the different physical forces that stabilise the native form of lysozyme are unchanged after SEDS processing. From FT‐Raman spectroscopic studies secondary structural changes were observed in certain SEDS‐produced lysozyme, with most processed samples displaying a slightly more disordered secondary structure than the unprocessed sample. However, SEDS samples produced at 200 bar and 40 °C exhibited negligible disturbance. Thus the SEDS process utilising aqueous solution was able to bring about size reduction of lysozyme with minimal loss of biological activity.

A discriminatory intrinsic dissolution study using UV area imaging analysis to gain additional insights into the dissolution behaviour of active pharmaceutical ingredients

For efficient and effective drug development it is desirable to acquire a deep understanding of the dissolution behaviour of potential candidate drugs and their physical forms as early as possible and with the limited amounts of material that are available at that time. Using 3-10mg sample quantities, the ability of a UV imaging system is investigated to provide deep mechanistic insight into the intrinsic dissolution profiling of a range of compounds and physical forms assessed under flow conditions. Physical forms of indomethacin, theophylline and ibuprofen were compressed and their solid-state form confirmed before and after compression with X-ray methods and/or Raman spectroscopy. Intrinsic dissolution rates (IDRs) were determined using the compacts UV-imaging profile. The ratio in the IDRs for theophylline anhydrate over hydrate was 2.1 and the ratio for the alpha form of indomethacin over the gamma form was approximately 1.7. The discriminatory power of the novel UV area visualisation approach was shown to be high in that process-induced solid-state dissolution differences post-micronisation could be detected. Additionally, the scale-down system was able to visualise a previously observed increase in ibuprofen IDR with an increase in concentration of sodium dodecyl sulphate. The mechanistic dissolution insights from the visualisation approach are evident.