Determination of total protein in highly purified factor IX concentrates

Protein determination by the methods of Kjeldahl, Biuret, Bradford and UV absorbance at 280 nm have been studied in regard to accuracy, precision and simplicity. A reference preparation of a highly purified factor IX concentrate, Nanotiv, reconstituted to 1/5 of ordinary volume was used in the study in order to make a comparison between the different procedures. The Kjeldahl method resulted in a protein concentration of 3.7 mg/ml, whereas the Biuret, Bradford (BSA) and UV absorbance at 280 nm resulted in protein concentrations of 3.6, 2.5 and 2.8 mg/ml, respectively. The corresponding values for specific activity were 136, 140, 200 and 179 IU/mg, respectively. These results demonstrate a great variation in the response obtained by different methods for determination of total protein.