Wei Wang1, Drew N Kelner. 1. Analytics & Formulation, Process Sciences, Biotechnology, Bayer Corp., 800 Dwight Way, Berkeley, California 94701, USA. wei.wang.b@bayer.com
Abstract
PURPOSE: This study was designed to investigate the stability of recombinant FVIII (rFVIII) in solution at different pHs and to probe the cause(s) of rFVIII inactivation under accelerated storage conditions. METHODS: Aqueous stability samples of full-length rFVIII at different pHs were incubated at 40 degrees C for several days and analyzed by the one-stage clotting assay. SEC-HPLC, SDS-PAGE, and UV spectrophotometry. RESULTS: Incubation of liquid rFVIII at 40 degrees C inactivated the protein rapidly and linearly with time on a semi-log scale at all pHs, suggesting a first order or pseudo first order process. A U-shaped relationship was found between the rate constant for loss of rFVIII activity and the solution pH. The minimal rate of inactivation was found between pH 6.6 and 7.0 with a half-life of approximately 4 days. The SEC-HPLC results indicated pH-dependent aggregation of rFVIII during incubation. It was found that the disappearance of monomeric rFVIII by SEC-HPLC correlated with the loss of rFVIII activity (r2 = 0.97). Both the SDS-PAGE and UV results confirmed the aggregation pathway of rFVIII. In addition, the SDS-PAGE results suggest involvement of three aggregation mechanisms--disulfide-bond formation/exchange, non-reducible crosslinking, and physical interactions. CONCLUSIONS: The full-length rFVIII is unstable in solution at 40 degrees C and loses activity rapidly through a first order or pseudo first order aggregation process, which consists of both physical and chemical pathways. SEC-HPLC may be used in monitoring rFVIII stability studies in lieu of the clotting assay under the incubation conditions used in this study.
PURPOSE: This study was designed to investigate the stability of recombinant FVIII (rFVIII) in solution at different pHs and to probe the cause(s) of rFVIII inactivation under accelerated storage conditions. METHODS: Aqueous stability samples of full-length rFVIII at different pHs were incubated at 40 degrees C for several days and analyzed by the one-stage clotting assay. SEC-HPLC, SDS-PAGE, and UV spectrophotometry. RESULTS: Incubation of liquid rFVIII at 40 degrees C inactivated the protein rapidly and linearly with time on a semi-log scale at all pHs, suggesting a first order or pseudo first order process. A U-shaped relationship was found between the rate constant for loss of rFVIII activity and the solution pH. The minimal rate of inactivation was found between pH 6.6 and 7.0 with a half-life of approximately 4 days. The SEC-HPLC results indicated pH-dependent aggregation of rFVIII during incubation. It was found that the disappearance of monomeric rFVIII by SEC-HPLC correlated with the loss of rFVIII activity (r2 = 0.97). Both the SDS-PAGE and UV results confirmed the aggregation pathway of rFVIII. In addition, the SDS-PAGE results suggest involvement of three aggregation mechanisms--disulfide-bond formation/exchange, non-reducible crosslinking, and physical interactions. CONCLUSIONS: The full-length rFVIII is unstable in solution at 40 degrees C and loses activity rapidly through a first order or pseudo first order aggregation process, which consists of both physical and chemical pathways. SEC-HPLC may be used in monitoring rFVIII stability studies in lieu of the clotting assay under the incubation conditions used in this study.
Authors: Karthik Ramani; Vivek S Purohit; Razvan D Miclea; C Russell Middaugh; Sathyamangalam V Balasubramanian Journal: J Pharm Sci Date: 2005-06 Impact factor: 3.534
Authors: Marisa K Joubert; Martha Hokom; Catherine Eakin; Lei Zhou; Meghana Deshpande; Matthew P Baker; Theresa J Goletz; Bruce A Kerwin; Naren Chirmule; Linda O Narhi; Vibha Jawa Journal: J Biol Chem Date: 2012-05-14 Impact factor: 5.157