Valeska P.
Ting
a,
Marc
Schmidtmann
b,
Paul F.
Henry
c,
Sandie E.
Dann
d,
Jenna L.
Crisp
d,
Chick C.
Wilson
b and
Mark T.
Weller
*a
aSchool of Chemistry, University of Southampton, U.K.. E-mail: mtw@soton.ac.uk; Fax: +44 23 8059 33781; Tel: +44 23 8059 3592
bWestCHEM, Department of Chemistry, University of Glasgow, U.K.. E-mail: c.c.wilson@chem.gla.ac.uk; Tel: +44 141 330 8522
cMI-1, Helmholtz Zentrum Berlin für Materialien und Energie, Berlin, Germany. E-mail: paul.henry@helmholtz-berlin.de; Tel: +49 30 8062 2686
dDepartment of Chemistry, Loughborough University, U.K.. E-mail: s.e.dann@lboro.ac.uk
First published on 11th October 2010
The hydrations of the excipients stable α-lactose (Lαs), with two particle sizes, and trehalose have been studied by neutron powder diffraction; analysis of the Avrami plots, derived from the bulk polycrystalline phase compositions, allows elucidation of the kinetics and mechanisms involved in these hydration processes.
Trehalose is used as a protein-stabilizing agent and is used in several biopharmaceutical monoclonal antibody formulations, in organ protection solutions and as a moisturizer in cosmetics and dry eye treatments. It is also described as a potential excipient for use in powder inhalers. The significance of studying the behaviours of (bio)pharmaceutical compounds, particularly in situ during thermal or humidity changes, has been recently discussed by a number of authors.11–14 Information on phase stability and phase transformations as a result of heating, cooling, or water or solvent molecule uptake is relevant due to ambient weather conditions in various countries where DPI units are used. However, the changes in relative humidity can be obtained using powder X-ray diffraction, including structural changes that occur. Powder X-ray diffraction has inherent drawbacks, as it samples mainly the surface of a polycrystalline mixture so that it is only structural changes occurring on the surface of a crystallite that are probed. This limitation is of particular concern when investigating the phase and reaction chemistry of a pharma-compound as a function of particle size – a key parameter in controlling dissolution rates.15–18 Similar limitations apply to other commonly-used techniques for studying pharmaceuticals in situ, such as infrared and Raman spectroscopy and optical microscopy, in that these methods are also confined to monitoring the surface reaction chemistry. Therefore, to determine changes in bulk and overall structure, and also to investigate transformation kinetics as a function of particle size, a method that probes the entire bulk sample is required. To this end we have used neutron diffraction, which is well known as a non-destructive technique for in situ investigations of bulk systems and non-surface structures, for example, of archaeological artefacts and engineering structures. Problems with incoherent scattering from the 1H isotope, which may have precluded application of this method to hydrogenous pharmaceutical compounds previously, have recently been overcome through use of modern high-flux instrumentation.19 Here, we describe the in situ hydration of α-lactose (Lαs) samples of different particle sizes (∼50 μm and >1 μm) and trehalose followed by neutron powder diffraction (NPD), demonstrating the type of information that can be extracted on the kinetics of hydration processes for bulk polycrystalline materials.
For the in situ hydration experiments, powdered samples of stable α-lactose (two samples, the highly crystalline form described above, and a small particle size form (particle size 0.1–1μm), hereafter denoted SPSα-lactose or trehalose (>99%) were loaded into 5 mm diameter containers formed from a heavily perforated COMPOUND LINKS
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Download mol file of compoundvanadium-foil. The can size was kept to a minimum to reduce the possibility of moisture gradients between powder at the surface and the core of the can while ensuring that diffraction data collected in 2.5 min intervals had sufficient peak-to-background ratios to permit profile refinement with extraction of phase fractions. The sample environment consisted of heated aluminium vessel with an atmosphere of controlled-humidity modified from that described previously.22 A regulated flow of humid gas was achieved through the use of a RH-200 humidity generator unit, which combined a stream of dry nitrogen gas with a humidified N2 stream in a mixing ratio that could be set and remotely controlled via a computer program. The humidity of the output gas stream was directly measured within the generator unit using a dew-point sensor and in the sample chamber using an in-cell hygrometer. Temperature control was achieved over the temperature range 30–200 °C through heating tapes mounted around the aluminium chamber and a remote COMPOUND LINKS
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Download mol file of compoundPID interface; pre–experiment tests showed that 100% humidity could be achieved in the cell at temperatures up to 55 °C and lower humidity values at the higher temperatures.
The background contribution from the humid gas flow through the sample environment was found to be negligible by measurements on a non-hygroscopic powder at various humidity levels, dispelling concerns of significant beam attenuation or incoherent scattering by the H2O vapour.
Full powder diffraction patterns were collected at 2.5 min intervals during the hydration of the HCα-lactose, SPSα-lactose and trehalose, under conditions of 100% RH at 25 °C. Diffraction profiles were modelled via Rietveld refinement as stated above; for the trehalose pairs of diffraction data sets were summed to give the equivalent of 5 min data sets. Analysis was undertaken using the extracted crystallographic models for stable α-lactose and α-lactose monohydrate (ESI†) and the literature data for, trehalose and trehalose dihydrate.2,3,7,9 During profile fitting only the profile parameters and the phase fractions of the anhydrous and hydrated disaccharides were refined. Sequential GSAS refinements (SEQGSAS)19 was used to analyse the full set of diffraction profiles for each system and the phase fraction ratio was extracted in each case.
X = 1 − e−(kt)n |
ln[−ln(1 − X)] = nlnk + nlnt |
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Fig. 1 Stacked plot viewed down the intensity direction showing the evolution of the diffraction patterns of standard stable α-lactose as a function of time in 100% relative humidity at 45 °C; the positions of three strong reflections from α-lactose monohydrate are shown. |
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Fig. 2 Phase percentage of α-lactose monohydrate in HCα-lactose (black filled circles) and SPSα-lactose (grey filled circles) exposed to 100% RH at 25 °C, as refined from sequential NPD data sets as a function of time. |
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Fig. 3 Avrami plots for the formation of α-lactose monohydrate from HCα-lactose (circles) and SPSα-lactose (triangles) exposed to a humid gas flow, as a function of time. |
For trehalose a similar analysis shows slightly more complex behaviour, Fig. 4 and 5. The phase fraction of dihydrate does not present a simple sigmoid shape but rather an initial induction period of around 60 min followed a more rapid growth of dihydrate finally a slower rate of formation of the product phase. A straight line fit to the Avrami plot is much poorer (r2 = 0.984) though it yields a similar value for n (1.645) as was found for stable α-lactose. A better fit to this plot can be achieved using two straight lines corresponding to a reaction time up to 150 min (n = 2.35, r2 = 0.973) and thereafter (n = 1.45, r2 = 0.995. This behaviour might be explained by the fact that the process of hydration of trehalose eventually leads to trehalose dihydrate and probably involves a two stage mechanism with an intermediate partially hydrated phase. As no additional unfitted reflection was observed at intermediate hydration times it seems likely that only small levels of this intermediate phase are formed at the reaction boundary or that it is amorphous. The formation of this intermediate phase may account for the induction time before the dihydrate phase is observed. The values of n for the two reaction processes, between 2–3 and 1–2 respectively, are consistent with an initial 3-dimensional bulk growth process for the formation of the initial intermediate phase followed by a lower dimensionality (i.e. surface or linear) growth for crystallisation of the dihydrate.
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Fig. 4 Phase percent of trehalose dihydrate as a function of time, as refined from sequential NPD data from anhydrous trehalose exposed to 100% RH. at 25 °C. |
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Fig. 5 Avrami plot for the hydration of trehalose; linear regression fitted to whole data range is shown as solid grey line. Fits to data at short reaction times and long reaction times are shown as grey dash-dot and black dashed lines respectively. |
The ability to observe the structures of pharmaceutical compounds in situ in controlled humidity and temperature conditions and study the bulk material rather than simply the surface structures is of considerable relevance to production and the storage of many active pharmaceutical compounds. The technique could also readily be applied to complex formulations and those in pelletized forms.
Footnote |
† Electronic supplementary information (ESI) available: Supplementary figures and final crystallographic model. See DOI: 10.1039/c0md00093k |
This journal is © The Royal Society of Chemistry 2010 |