Protein Quantity on the Air–Solid Interface Determines Degradation Rates of Human Growth Hormone in Lyophilized Samples

Abstract

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.

Formation of Stable Nanobubbles on Reconstituting Lyophilized Formulations Containing Trehalose

“…13 After holding the samples at À45 C for 400 min, the shelf temperature was increased to À5 C during 30 min and held at À5 C for 6 h. Then the shelf temperature was reduced to À45 C at 1.3 C/min and held at this temperature for 6 h.…”

Heterogeneity of Protein Environments in Frozen Solutions and in the Dried State

“…Also, note that crystallization of a solute (such as buffer or lyoprotector) would be an additional source of a heterogeneity, solute crystallization and its relevance to stability of frozen and freeze-dried formulations was discussed elsewhere [5,10,11,64] and not considered here. Difference in the stability of different populations of proteins was convincingly demonstrated for the case of proteins on air-solid interface in freeze-dried cakes, [31] with the degradation rates on the interface exceeding the rate of bulk molecules by at least two orders of magnitude. As a result of such heterogeneity, shelf life of a pharmaceutical protein formulation would be limited by the most unstable population of protein molecules.…”

“…It was shown that concentration of albumin in dimethyl sulfoxide (DMSO)/water solutions was high at the ice interface at low temperatures and as much as 20 % of the albumin (for 32-53 mg/mL solutions) can be adsorbed on the ice or entrapped in the ice phase. In a recent study of the freezedried recombinant human growth hormone (rhGH) [31], the amount of protein on the surface of the freeze-dried cake was determined using electron spectroscopy for chemical analysis in formulations with sucrose, trehalose and hydroxyethyl starch (HES). The freeze-dried formulations were prepared at five different freezing conditions that include standard lyophilization cycle with slow freezing, pre-annealing before primary drying, post-annealing after secondary drying, fast freezing by immersion of vials into liquid nitrogen, and fast freezing of droplets by pipetting solution into immersed in liquid nitrogen vial.…”

Reduced Subvisible Particle Formation in Lyophilized Intravenous Immunoglobulin Formulations Containing Polysorbate 20

“…9,12,[17][18][19] After sublimation of ice crystals during primary drying, these protein molecules would be found at the remaining glassy solid-air interface. 9,20,21 In 2 recent studies, the extent of damage (oxidation, deamidation, and aggregation) to 2 therapeutic proteins observed after long-term storage correlated directly with the amount of protein exposed on the glassy solid-air interface. 20,21 Annealing the frozen formulations prior to drying reduced the specific surface areas (SSAs) of the glassy solid and the fraction of protein exposed at the solid-air interface, which in turn significantly reduced degradation of the protein.…”

“…9,20,21 In 2 recent studies, the extent of damage (oxidation, deamidation, and aggregation) to 2 therapeutic proteins observed after long-term storage correlated directly with the amount of protein exposed on the glassy solid-air interface. 20,21 Annealing the frozen formulations prior to drying reduced the specific surface areas (SSAs) of the glassy solid and the fraction of protein exposed at the solid-air interface, which in turn significantly reduced degradation of the protein. 21 The first goal of this study was to test the hypothesis that conditions that result in lower amounts of a model antibody therapeutic on the surface of glassy solids would in turn reduce aggregation and particle formation rates during storage.…”