Michael L. Roy

The Effects of Formulation Variables on the Stability of Freeze-Dried Human Growth Hormone

Formulation often has a dramatic effect on degradation of proteins during the freeze-drying process as well as impacting on the “shelf-life” stability of the freeze-dried product. This research presents the results of a formulation optimization study of the “in-process” and shelf-life stability of freeze-dried human growth hormone (hGH). Chemical decomposition via methionine oxidation and deamidation of asparagine residues as well as irreversible aggregation were characterized by HPLC assay methodology. In-process degradation and stability of low moisture freeze-dried solids were studied at 25 and 40°C in a nominal nitrogen headspace (≈0.5% O2). Formulation variables included pH, level of salts, and the nature of the lyoprotectant. Studies of the effect of shear on aggregation in solutions indicated that shear comparable to that experienced during filtration does not induce aggregation. Irreversible changes in hGH during the freeze-drying process were minimal, but chemical decomposition via methionine oxidation and asparagine deamidation and aggregation did occur on storage of the freeze-dried solid. Decomposition via methionine oxidation was significant. A combination of mannitol and glycine, where the glycine remains amorphous, provided the greatest protection against decomposition and aggregation. It is postulated that an excipient system that remains at least partially amorphous is necessary for stabilization. However, the observation that dextran 40 formulations showed poor stability toward aggregation demonstrates that an amorphous excipient system is not a sufficient condition for stability. Stability of the solid was optimal when produced from solutions in the pH range, 7–7.5, with severe aggregation being observed at high pH. The level of sodium phosphate buffer affected stability of the solid, although this relationship was complex. Freeze-drying in the presence of NaCl produced severe aggregation and precipitation during the freeze-drying process as well as acceleration of oxidation and/or deamidation.

The secondary drying stage of freeze drying : drying kinetics as a function of temperature and chamber pressure

Abstract Secondary drying involves removal of water which did not freeze. This report emphasizes the phenomenological description of the effects of temperature and chamber pressure on the kinetics of drying. A crystalline solute (mannitol) and two amorphous solutes (moxalactam di-sodium and povidone) were selected for study. Drying kinetics were determined gravimetrically using a vacuum microbalance and by Karl Fischer assay of vials sealed at selected times during secondary drying experiments conducted in a laboratory scale freeze dryer. The main observations may be summarized as follows: (1) the water content decreases rapidly during the first few hours of drying and then appears to approach a plateau level of residual water which far exceeds the equilibrium water content calculated from the desorption isotherm data and the measured partial pressure of water in the drying chamber; (2) this plateau level of water sharply decreases as the drying temperature is increased; (3) the drying rate increases as the product specific surface area increases; and (4) variations in chamber pressure (0–0.2 mmHg) and dried product thickness have little or no effect on drying rate. We conclude that the rate-limiting mass transfer process for drying an amorphous solid is either evaporation at the solid/vapor interface or diffusion in the solid, probably the former. The ‘plateau level kinetics’ appears to be consistent with amorphous particle size heterogeneity superimposed on a simple model based on Fickian diffusion with rate controlling surface evaporation.

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.