Development of freeze-dried biosynthetic Factor VIII: I. A case study in the optimization of formulation

Abstract

The purpose of this paper was to identify an optimal formulation, free of any human or animal derived protein, which stabilizes biosynthetic Factor VIII (rAHF) during freeze drying and storage. Factor VIII activity in samples stored at temperatures between 25 degrees C and 60 degrees C was determined using the one-stage activated partial thromboplastin time assay. Various formulations containing different combinations of a stabilizer and a bulking agent were screened for acceptable freeze-drying behavior, elegance of the resulting product, and stability during processing and storage. Degradation of freeze-dried rAHF followed the 'square root of time' kinetics. Stability of rAHF was found to increase with increasing protein concentration, indicating a self-protection effect. The addition of the antioxidant, glutathione was also shown to enhance storage stability. Given the constraint of high residual levels of NaCl from purification, the lead formulation employed mannitol as a bulking agent and trehalose as the general stabilizer. This formulation allowed an elegant product to be produced which more than met the stability requirements. However, it was also shown that elimination of residual NaCl allowed a much shorter freeze-drying cycle to produce an elegant product with greatly enhanced stability.

Room temperature stabilization of oral, live attenuated Salmonella enterica serovar Typhi-vectored vaccines

“…The increased osmolarity of the growth media may allow Ty21a to develop tolerance to osmotic stress, which would be encountered during desiccation. The higher titer of bacteria at stationary phase may benefit from a similar self-protective mechanism to desiccation encountered by proteins at high concentrations [59]. …”

Photolysis of Recombinant Human Insulin in the Solid State: Formation of a Dithiohemiacetal Product at the C-Terminal Disulfide Bond

“…The important question is whether such products are also formed during photoirradiation of protein solids. Some common degradation products of protein solids have been documented (25)(26)(27)(28). However, the photodegradation of solid proteins has only been studied in a few cases (29,30).…”

Addition of Monovalent Electrolytes to Improve Storage Stability of Freeze-Dried Protein Formulations

“…[2][3][4] However, addition of other components to the disaccharide-based formulation has also been investigated, such as polyols, polymers, and amino acids. [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] Nominally, polymers would seem to be good stabilizer candidates because they increase the glass transition temperature (T g ). 1,[6][7][8] However, whether T g is a reliable predictor of stability well below T g is doubtful.…”

“…17 Electrolytes are relatively common in freeze-dried formulations, as buffers and often as NaCl arising from prior purification steps. 9 However, because NaCl and buffer salts depress the collapse temperature, 11 use of salts in formulations is normally restricted to very low levels. In this study, we investigate addition of alkali chlorides, such as sodium chloride, as stability enhancers in sucrose-based formulations.…”