Characterization of aggregates of recombinant human factor VIII by size-exclusion chromatography and immunoassay

The presence of aggregates of Factor VIII (FVIII) was assessed for a highly purified form (rFVIII) and for the formulated preparation containing human serum albumin (rFVIIIf). Size-exclusion chromatography (SEC) on Sepharose CL-6B and TSK 4000 matrices under native conditions revealed < 1% aggregates of FVIII in either rFVIII or rFVIIIf, as determined by FVIII immunoassay of chromatographic fractions. No degradation products were observed. The immunoassay was capable of detecting FVIII immunoreactivity to levels of approximately 5 ng/ml. Overlap of FVIII clotting (FVIII:c) and immunoassay (FVIII:cAg) activities was observed for SEC fractions. Heat stressing of rFVIIIf (47.5 degrees C for 24 h) resulted in a quantitative increase in FVIII-positive material in the void volume of a TSK 4000 column, demonstrating that aggregates of FVIII can be produced and detected by this method. We conclude that aggregates of FVIII in rFVIII and rFVIIIf constitute < or = 1% by the method described.