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rhGH was lyophilized with various glass-forming stabilizers, employing cycles that incorporated various freezing and annealing procedures to manipulate glass formation kinetics, associated relaxation processes and glass specific surface areas (SSA’s). The secondary structure in the cake was monitored by IR and in reconstituted samples by CD. The rhGH concentrations on the surface of lyophilized powders were determined from ESCA. Tg, SSA’s and water contents were determined immediately after lyophilization. Lyophilized samples were incubated at 323 K for 16 weeks, and the resulting extents of rhGH aggregation, oxidation and deamidation were determined after rehydration. Water contents and Tg were independent of lyophilization process parameters. Compared to samples lyophilized after rapid freezing, rhGH in samples that had been annealed in frozen solids prior to drying, or annealed in glassy solids after secondary drying retained more native-like protein secondary structure, had a smaller fraction of the protein on the surface of the cake and exhibited lower levels of degradation during incubation. A simple kinetic model suggested that the differences in the extent of rhGH degradation during storage in the dried state between different formulations and processing methods could largely be ascribed to the associated levels of rhGH at the solid-air interface after lyophilization.

Lyophilized formulations of keratinocyte growth factor-2 (KGF-2) were prepared with a range of disaccharide (sucrose or trehalose) and hydroxyethyl starch (HES) mass ratios. Protein degradation was assessed as a function of time of storage of the dried formulations at 40, 50 and 60 °C. Lyophilized and stored samples were rehydrated, and protein degradation was quantified by measuring loss of monomeric protein with size exclusion chromatography and by determining chemical degradation in the soluble fraction with reverse-phase chromatography. The secondary structure of the protein in the lyophilized formulations was studied with infrared spectroscopy. The magnitudes of degradation were compared the key physical properties of the formulations including retention of protein native secondary structure, glass transition temperature (Tg), inverse mean square displacements −1 for hydrogen atoms (fast β relaxation), and the relaxation time τβ, which correlates with relaxation due to fast Johari-Goldstein motions in the glass[1]. In addition, specific surface areas of the lyophilized formulations were determined by Brunauer-Emmet-Teller analysis of krypton adsorption isotherms and used to estimate the fraction of the KGF-2 molecules residing at the solid-air interface. KGF-2 degradation rates were highest in formulations wherein the protein’s structure was most perturbed, and wherein β relaxations were fastest, but the dominant factor governing KGF-2 degradation in freeze-dried formulations was the fraction of the protein found at the glass solid-air interface.

Because aqueous liposomal formulations containing multiply unsaturated lipids are susceptible to chemical degradation, these formulations are often lyophilized. Despite their limited chemical stability, interest in the use of multiply unsaturated lipids to promote intracellular delivery has increased considerably in recent years. The goal of the current study was to examine the long term storage stability of lyophilized formulations containing lipids with increasing levels of unsaturation, and various strategies which can be employed to improve stability. Aqueous lipid-trehalose formulations containing 1,2-dilinolenoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLinPC) or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) were lyophilized and stored at temperatures ranging from 4°C to 60°C. We observed that the lipid degradation rate increased as the storage temperature and unsaturation level were increased. Even the cleanest sugars which are available commercially contain iron contaminants, and it was observed that the chelation of these iron contaminants significantly improved the stability of DLPC during storage. However, the glass transition temperature of the sugar which was included in the formulation, the reduction of the oxygen in the aqueous sample prior to lyophilization, the inclusion of helper lipids (i.e., cholesterol), and the rate of freezing did not significantly improve stability.

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