Solid state stability of proteins III: calorimetric (DSC) and spectroscopic (FTIR) characterization of thermal denaturation in freeze dried human growth hormone (hGH).

This research is a study of the changes in secondary structure (Fourier transform infrared spectroscopy, FTIR), aggregation, and loss of the magnitude of the heat of denaturation upon scanning to and partially through the temperature range of the thermal denaturation peak of a model protein, human growth hormone (hGH). We study two formulations, a system of essentially pure protein (with a trace of phosphate buffer) and a system formulated with trehalose in a 3:1 trehalose:hGH weight ratio. The extent of denaturation is measured by loss of secondary structure by FTIR, the loss of heat of denaturation by differential scanning calorimetry (DSC), and the fraction of protein aggregated by HPLC. We examine loss of structure on heating to the DSC onset of thermal denaturation and restoration of structure by cooling below the denaturation temperature and holding to (nominally) allow time for refolding, and we also examine restoration of structure upon dissolving and refreeze drying samples heated to selected temperatures in the denaturation range. We find that denaturation occurs only above the glass transition temperature, is highly cooperative, and is only reversible by redissolving the "denatured" formulated (trehalose) solid. Further, all measures of the extent of denaturation are in essential agreement.

Solid state chemistry of proteins: II. The correlation of storage stability of freeze-dried human growth hormone (hGH) with structure and dynamics in the glassy solid.

This research presents storage stability of human growth hormone, hGH, in lyophilized di-saccharide formulations. Stability via HPLC assay was assessed at 40 and 50 degrees C. Structure of the protein in the solids was assessed by infrared spectroscopy. Molecular mobility was characterized by structural relaxation times estimated from DSC data and by measurement of atomic motion on a nanosecond time scale by neutron scattering. Very large stability differences were observed among the various formulations, with both chemical and aggregation stability showing the same qualitative trends with formulation. Near the T(g), T(g) appeared to be a relevant stability parameter, but for storage well below T(g), stability seems unrelated to T(g). Stability (chemical and aggregation) was weakly correlated with secondary structure of the protein, and there was a partial quantitative correlation between degradation rate and the structural relaxation time. However, at equivalent levels of disaccharide relative to protein, sucrose systems were about a factor of two more stable than trehalose formulations, but yet had greater mobility as measured by structural relaxation time. Secondary structure was equivalent in both formulations. Neutron scattering results documented greater suppression of fast dynamics by sucrose than by trehalose, suggesting that well below T(g), fast dynamics are important to stability.

Biophysical signatures of noncovalent aggregates formed by a glucagonlike peptide-1 analog: a prototypical example of biopharmaceutical aggregation.

LY307161 is a 31 amino acid analog of glucagonlike peptide-1(7-37)OH susceptible to physical instability associated with pharmaceutical processing. Orthogonal biophysical studies were conducted to explore the origins of this physical instability and to distinguish pharmaceutically desirable states of this aggregating peptide from undesirable ones. Equilibrium sedimentation analysis established that LY307161 exists as a monomer at pH 3, and reversibly self-associates in the pH range 7.5-10.5. Causative factors for physical instability related to lyophilization conditions were investigated. Solution pH, acetonitrile content, and concentration of the peptide prior to lyophilization each impacted physicochemical properties of the resultant powders. A comparative study of two powder samples exhibiting physicochemically disparate properties established that LY307161 forms soluble noncovalent aggregates. FT-IR analyses in the solid and solution states identified a prominent band at 1657-1659 cm(-1) attributed to alpha-helix structure. Noncovalent soluble aggregate exhibited characteristic bands at 1615 and 1698 cm(-1) indicative of intermolecular beta-sheet structure. An agitation-induced, precipitated solid form of LY307161 exhibited a different FT-IR signature indicative of a conformationally distinct species. Circular dichroism and fluorescence spectroscopy, together with dynamic light scattering measurements and dye-aggregate complexation, provided additional insights into the distinctions between aggregated and native LY307161.

Freeze-drying of tert-butanol/water cosolvent systems: a case report on formation of a friable freeze-dried powder of tobramycin sulfate.

A case study is presented in which a tert-butanol (TBA)/water cosolvent system was found to be a useful means of producing freeze-dried tobramycin sulfate that readily forms a loose powder upon agitation in a specialized application in which a critical quality attribute is the ability to pour the sterile powder from the vial. Both formulation and processing variables are important in achieving acceptable physical properties of the cake as well as minimizing residual TBA levels. Liquid/liquid phase separation was observed above critical concentrations of both drug and TBA, resulting in a two-layered lyophilized cake with unacceptable appearance, physical properties, and residual TBA levels. However, the choice of tobramycin sulfate and TBA concentrations in the single-phase region of the phase diagram resulted in a lyophilized solid that can readily be poured from vials. Crystallization of TBA before drying is critical to achieving adequately low residual TBA levels, and this is reflected in the effect of thermal history of freezing on residual TBA levels, where rapid freezing results in incomplete crystallization of TBA and relatively high levels of residual solvent. Annealing at a temperature above T'(g) of the system after an initial freezing step significantly reduces the level of residual TBA. Secondary drying, even at increased temperature and for extended times, is not an effective method of reducing residual TBA levels.