Elsevier

Cryobiology

Volume 61, Issue 1, August 2010, Pages 27-32
Cryobiology

Use of dynamic mechanical analysis (DMA) to determine critical transition temperatures in frozen biomaterials intended for lyophilization

https://doi.org/10.1016/j.cryobiol.2010.04.002Get rights and content

Abstract

Dynamic mechanical analysis is widely used to determine glass transitions in solid state materials. However, here we demonstrate the application of DMA for the determination of glass transitions (Tg) in the frozen liquid state by means of a steel sample pocket. The use of the pocket allows frozen material to be analysed and glass transition events demonstrated. In addition, it allows weak glass transitions to be detected clearly in some complex formulations where they can be obscured by eutectic and other strong thermal events when other methods such as DSC or DTA are used. Classical excipients (trehalose, lactose, dextran) were analysed and shown to give reproducible Tg values, though with values slightly higher than those obtained by DSC. Finally, several complex real biological materials, typical of those encountered when freeze drying biological and biopharmaceutical materials, were analysed and the potential value of DMA demonstrated to determine the relevant glass transition temperatures for use in cryobiology and freeze drying.

Introduction

Dynamic mechanical (thermal) analysis (DMA or DMTA) measures phase morphology transitions (melts, crystallizations, alpha transitions (glass) and beta transitions) of materials by vibrating the material sinusoidally at a constant frequency and low amplitude and monitoring the stiffness and damping with respect to temperature [15]. DMA has historically been used to determine the Tg (glass transitions) in solid polymers and co-polymers [6], [8], [14], in food materials [1] and pharmaceuticals [9]. Recent application of material pockets – designed originally to allow analysis of 10–30 mg of powder in the thin stainless steel clamp [5], [17] – has shown that a drop of liquid that wets the steel can be frozen in situ and then reheated, revealing any melt (Tm) and/or glass transition (Tg) events. In the past filter papers wetted with liquid have been frozen in the DMA clamps, but then one has to carefully adjust for the tan delta contribution from the coated paper elements. Steel has no such damping peaks in the −100 °C to +100 °C region of interest when analyzing biological materials.

Differential scanning calorimetry (DSC) or modulated differential scanning calorimetry (mDSC) has been used for many years but often has difficulty in detecting Tg’s in some materials due to the presence of much greater melt or eutectic events, and this has given rise to the claim that DMA can often be one thousand times more sensitive for determination of Tg′ in samples where there are only trace amount of polymers present – for example in composite or co-polymer materials. Resolution of the Tg by DMA can be shown in powder samples with known transitions on less than 1 mg held in steel clamps of 700 mg (with 100 times the stiffness of the tested material) indicative of the inherent sensitivity of the method. Furthermore by multiplexing frequencies (e.g. at 1 Hz and 10 Hz, or 0.3, 3 and 30 Hz) during a thermal scan it is possible to distinguish between the Tg′, which is frequency dependent, and the Tm which happens independently of the vibration frequency. Activation energies can also be calculated for the transition using an Arrhenius plot. Royall et al. [17] described the use of these pockets when analyzing dry lactose powders by DMA and showed its utility in determining the amorphous content of the solid.

Lyophilization (freeze drying) is widely used in the biopharmaceutical industry for stabilizing labile biomolecules [2], by removing the greater proportion of the water present to leave behind a dried cake which rapidly and fully reconstitutes on addition of resuspending medium. When determining lyophilization conditions it is vital to identify the glass transition (Tg′) temperature in the frozen state which indicates when the maximally concentrated amorphous state has been achieved [12]. During primary drying, while the majority of water is being removed by sublimation, this temperature should not be exceeded in the product if structural collapse of the cake is to be avoided and a pharmaceutically elegant appearance is to be maintained [13], [19].

Commonly used techniques for determining subambient thermal events, such as eutectic points or glass transitions, include modulated differential scanning calorimetry [10], differential thermal analysis [16] and freeze drying microscopy [7], [12], each of these may be best suited to particular formulations. In this communication we demonstrate the use of DMA in the determination of glass transitions and have evaluated DMA with a number of biological materials to identify glass transition events.

Section snippets

Materials

Biological reference materials were obtained from the NIBSC catalogue (<www.nibsc.ac.uk/products/html>). These materials were prepared at NIBSC in lyophilized format and reconstituted just before analysis.

Lyophilized trehalose (98/622) 10 mg per ampoule, reconstituted typically to 10 mg/ml in distilled water – this disaccharide is a popular stabilizer choice for the lyophilization of labile biological materials.

Heparin (05-029-PM) 20 mg per ampoule, reconstituted to 20–40 mg/ml in distilled water –

Determination of glass transition in the frozen state

In order to show that the Tan δ changes observed were due to the glass transition of the excipients or biologicals, and not other thermal events, frozen deionised water was studied and shown to give a broad spectrum with a tan delta around 2 °C. In most of the samples subsequently studied the water signal was not a major influence. Similarly, a sample of a purely crystalline material (5% w/v sodium chloride, eutectic point (Teu) = −21.5 °C) was examined. This showed a eutectic event, but no glass

Discussion

DMA can be used to determine glass transitions in common lyoprotectant excipients (lactose, trehalose, dextran) in the frozen state using a steel powder pouch to contain the sample. Several vibration frequencies were studied, since for simpler systems it is possible to differentiate between glass transitions (Tg) and crystalline melts by multiplexing a number of frequencies; the lower the frequency generally the sharper the peak in Tan δ. Phase morphology changes such as Tg′/Tg are frequency

Acknowledgments

We thank the Cavendish Laboratory, University of Cambridge for providing laboratory facilities to JG.

References (21)

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