Heparin interference in the Bradford protein quantification assay

Abstract

Protein quantification in biological samples is a routine assay used in the biopharmaceutical industry during the purification procedures of plasma-derived products and/or recombinant proteins. Thus, choosing the method for this purpose is a critical step to obtaining reliable results. In this work, it was shown that Heparin (Hep), a compound commonly used as an excipient in the formulation step of Coagulation Factor Concentrates (CFC), interferes in protein quantification by the Bradford assay. This interference was proved both in solutions of Hep-fortified Bovine Serum Albumin (BSA) and in CFC samples. For a 40 mg % BSA solution, the Hep concentration which shows no interference in protein quantification is 1.6 IU/ml. It should be noted that a specific analysis must be carried out if working at a lower protein concentration, with a different protein, or with complex protein solutions, so the values obtained in this work can be used as a reference. Alternatively, Hep neutralization with Polybrene (Poly) was evaluated before protein determination by Bradford. In the case of BSA solutions, the interference was reversed and correct protein quantification was achieved, whereas in the CFC samples, the results were unsatisfactory. Until now, Hep interference in the Bradford assay has not been described in the scientific literature, nor listed in the inserts of the commercial kits for protein quantification by this method. It was concluded that for protein samples containing Hep, the choice of an alternative method, such as the one that uses bicinchoninic acid, is the most convenient option to obtain reliable results.