Assessment of Degree of Disorder (Amorphicity) of Lyophilized Formulations of Growth Hormone Using Isothermal Microcalorimetry

Abstract

When determining the degree of disorder of a lyophilized cake of a protein, it is important to use an appropriate analytical technique. Differential scanning calorimetry (DSC) and X‐ray powder diffraction (XRPD) are the most commonly used thermo‐analytical techniques for characterizing freeze‐dried protein formulations. Unfortunately, these methods are unable to detect solid‐state disorder at levels < 10%. Also, interpretation of DSC results for freeze‐dried protein formulations can be difficult, as a result of the more complex thermal events occurring with this technique. For example, proteins can inhibit the thermally induced recrystallization of the lyophilized cake, resulting in potential misinterpretation of DSC degree of disorder results. The aim of this investigation was to study the use of isothermal microcalorimetry (IMC) in the assessment of degree of solid‐state disorder (amorphicity) of lyophilized formulations of proteins. For this purpose, two formulations of growth hormone were prepared by lyophilization. These formulations consisted of the same amounts of protein, mannitol, glycine, and phosphate buffer, but differed in the freeze‐drying procedure. After lyophilization, the recrystallization of the samples was studied using IMC at 25°C under different relative humidities (58–75%). The effect of available surface area was studied by determining the heat of recrystallization (Q) of the samples before and after disintegration of the cakes. The results showed that, in contrast to DSC, IMC allowed detection of the recrystallization event in the formulations. Although both formulations were completely disordered and indistinguishable according to XRPD method, IMC revealed that formulation B had a different solid‐sate structure than formulation A. This difference was the result of differences in the freeze‐drying parameters, demonstrating the importance of choosing appropriate analytical methodology.