1. In-situ characterization of phase changes in lysozyme/ trehalose solutions using through vial impedance spectroscopy
M.S. Arshad, I. Ermolina ,E. Polygalov, G. Smith
Leicester School of Pharmacy, De Montfort University, Leicester. LE1 9BH  
INTRODUCTION
Lyophilization of a product which contains solutes with a propensity to form an amorphous state requires freezing and sublimation processes to be undertaken below the glass transition (Tg′) and /or collapse temperature in order to ensure a collapse-free stable matrix. These critical process parameters are measured off-line by differential scanning calorimetry (DSC) or freeze drying microscopy respectively. There are currently no methods for the in-process detection of these critical temperatures in such a way to enable the process development scientist to optimize the freezing cycle [1,2] . The present study aims to evaluate the application of in-vial impedance spectroscopy in monitoring phase changes in lysozyme/trehalose solutions formulation during freeze drying.
In three separate experiments, two 3 ml aliquots of each of the following solutions were transferred to bespoke impedance measurement vials (N=2) (5% w/v trehalose, 4.5% w/v Lysozyme (Sigma) and solution containing Lysozyme (4.5% w/v) with trehalose 1.5% w/v (3:1). The measurement vials were attached to a junction box located within the drying chamber of freeze drier HETO FD08 which connects to a high precision impedance analyser by means a hermetic pass-through. The impedance measurements were performed over a frequency range 100Hz-1MHz at scan interval of 1 min-1. The freezing cycle comprised the following steps; temperature hold at 25°C for 30 minutes, Temperature ramp from 25 °C to -35 °C during 60 minutes, temperature hold at -35 °C for 180 minutes and temperature ramp up from -35 °C to 25 °C during 120 minutes. The product temperature was also recorded in the neighbouring vial using thermocouple type K.
The complex capacitance spectrum records a step in the real part and a peak in the imaginary part. This process arises from the interfacial polarization of the product-electrode interface, and depends on the ionic concentration, and the temperature and viscosity of the formulation.
The interfacial process was characterized by an equivalent circuit comprising a constant phase element (CPE), a capacitor (C) and resistor (R).
Figure 1: Equivalent circuit to analyze the spectral data
Figure 2: Z-View fit results for individual spectrum
The results showed a number of non-linearities with temperature, during re-heating, suggesting possible multiple glass transitions.
Arrhenius plots of ln R vs. 1000/T displayed the typical non-linear behaviour, characteristic of glass forming liquids, with a suggestion that the strength of these glass forming solutions increased in the order lysozyme < lysozyme plus trehalose < trehalose.
Figure 3: Arrhenius plot of resistance (R ) element from Z-View fitting results during the freezing of trehalose and lysozyme solutions.
The derivative of the Arrhenius plot (Fig. 1) highlights another transition in behaviour close to – 17 °C, which may suggest a further change in the strength of each solution with increased temperature. Below this temperature, the strength of the glass forming liquid appears to follow the order lysozyme < lysozyme/trehalose mixture; whereas above this temperature the strength of the glass forming liquid followed the order lysozyme> lysozyme/trehalose mixture. Whether this observation is a result of phase separation remains to be seen, but it could point to a requirement for a specific temperature for the primary drying stage.
Figure 4: Derivative of the Arrhenius plot. The gradients of each line are indicative of the strength of the glass, with a greater gradient suggesting a weaker glass forming liquid.
It is speculated that while trehalose may provide a degree of protection through a generalized suppression of the molecular degrees of freedom (that might otherwise lead to instabilities of the protein e.g. unfolding and/or aggregation) this mechanism may be sensitive to the specific temperature at which the frozen solution is dried.
LyoDEA™ was developed through a collaboration with AstraZeneca, GEA Pharma Systems, and Ametek, and co-funded by the Technology Strategy Board .
[1]. Smith G, Polygalov E, Page T, inventors; GEA Pharma Systems Limited,A method for monitoring and/or controlling process parameters of a lyophilisation process. Great Britain patent GB /11/2011.
[2]. Smith G, Arshad MS, Polygalov E, Ermolina I. Eur J Pharm Biopharm. 2013;85(3 Pt B): Epub 2013/08/21.
MATERIALS AND METHODS
CONCLUSION
ACKNOWLEDGMENTS
REFERENCES
RESULTS AND DISCUSSION