Correlation of rFVIII inactivation with aggregation in solution

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.

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.

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.

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.