Conformational origin of the aggregation of recombinant human factor VIII

Aggregation of proteins is a major problem in their use as drugs and is also involved in a variety of pathological diseases. In this study, biophysical techniques were employed to investigate aggregate formation in the pharmaceutically important protein, recombinant human factor VIII (rhFVIII). Recombinant human factor Vm incubated in solution at 37 degreesC formed soluble aggregates as detected by molecular sieve chromatography and dynamic light scattering. This resulted in a corresponding loss of biological activity. Fluorescence and CD spectra of the thermally stressed rhFVIII samples did not, however, suggest significant differences in protein conformation. To identify conformational changes in rhFVIII that may be involved in rhFVIII aggregation, temperature and solutes were used to perturb the native structure of rhFVIII. Far-UV CD and FTIR studies of rhFVIII as a function of temperature revealed conformational changes corresponding to an increase in intermolecular beta -sheet content beginning at approximately 45 degreesC with significant aggregation observed above 60 degreesC. Fluorescence and DSC studies of rhFVIII also indicated conformational changes initiating between 45 and 50 degreesC. An increase in the exposure of hydrophobic surfaces was observed beginning at approximately 40 degreesC, as monitored by increased binding of the fluorescent probe, bis-anilinonaphthalene sulfonic acid (bis-ANS). perturbation by various solutes produced several transitions prior to extensive unfolding of rhFVIII. In all cases, a common transition, characterized by an increase in the wavelength of the fluorescence emission maximum of rhFVIII from approximately 330 to 335 nm, was observed during thermal and solute perturbation of factor VIII, Moreover, this transition was correlated with an increased association of factor VIII upon incubation at 37 degreesC in the presence of various solutes. These results suggest that association of rhFVIII in solution was initiated by a small transition in the tertiary structure of the protein which produced a nucleating species that led to the formation of inactive soluble aggregates.