title: 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.
authors.affiliation: Baxter Healthcare Corporation, Westlake Village, California, USA.
authors.forename: Feroz
authors.initials: F
authors.lastname: Jameel
authors.forename: Serguei
authors.initials: S
authors.lastname: Tchessalov
authors.forename: Erik
authors.initials: E
authors.lastname: Bjornson
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Lu
authors.forename: Marc
authors.initials: M
authors.lastname: Besman
authors.forename: Michael
authors.initials: M
authors.lastname: Pikal
mesh_terms: D000409:Alanine / Q000737:chemistry; D000975:Antioxidants / Q000737:chemistry; D001120:Arginine / Q000737:chemistry; D002152:Calorimetry, Differential Scanning; D002626:Chemistry, Pharmaceutical / Q000379:methods*; D004355:Drug Stability; D005169:Factor VIII / Q000096:biosynthesis* / Q000737:chemistry* / Q000378:metabolism; D005612:Freeze Drying / Q000379:methods; D005978:Glutathione / Q000737:chemistry; D005990:Glycerol / Q000737:chemistry; D005998:Glycine / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D006895:Hydroxyethyl Starch Derivatives / Q000737:chemistry; D007700:Kinetics; D008353:Mannitol / Q000737:chemistry; D010084:Oxidation-Reduction; D010314:Partial Thromboplastin Time; D055550:Protein Stability; D011887:Raffinose / Q000737:chemistry; D012965:Sodium Chloride / Q000737:chemistry; D013395:Sucrose / Q000737:chemistry; D013696:Temperature; D044366:Transition Temperature; D014199:Trehalose / Q000737:chemistry; D014961:X-Ray Diffraction
chemical_list: D000975:Antioxidants; D006895:Hydroxyethyl Starch Derivatives; D008353:Mannitol; D012965:Sodium Chloride; D006639:Histidine; D013395:Sucrose; C078147:F8 protein, human; D005169:Factor VIII; D001120:Arginine; D014199:Trehalose; D005978:Glutathione; D011887:Raffinose; D000409:Alanine; D005990:Glycerol; D005998:Glycine