aDepartment of Pharmacy and Biotechnology, Alma Mater Studiorum – University of Bologna, Via Belmeloro 6, 40126 Bologna, ItalybCenter for Applied Biomedical Research (CRBA), S. Orsola–Malpighi Hospital, Via Massarenti 9, 40138 Bologna, ItalycDepartment of Medical and Surgical Sciences, Alma Mater Studiorum – University of Bologna, Via Massarenti 9, 40138 Bologna, Italy
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The ADMET profile of drugs is strongly affected by human serum albumin (HSA), due to its leading role as carrier of poorly soluble compounds in plasma; a critical assessment of the binding capacity of HSA and the evaluation of binding competition between drugs are therefore pivotal for a reliable pharmacokinetic and pharmacodynamic characterization. In clinical practice, a potential source of impairment in the binding properties of HSA is the use of octanoate and N-acetyltryptophan as stabilizers during the production of pharmaceutical-grade HSA for infusion (i-HSA), which is currently administered in the treatment of a growing range of pathological conditions. The peculiar sensitivity of circular dichroism (CD) spectroscopy towards the stereochemical features of high-affinity binding events is herein exploited to achieve a site-specific assessment of the effect of stabilizers on the binding properties of i-HSA. The binding affinity and capacity of fatty-acid-free HSA towards site-selective induced circular dichroism (ICD) markers for the three high-affinity binding sites of HSA was compared to that of i-HSA submitted to ultrafiltration and dialysis to remove both stabilizers. Results showed a considerable impairment of the binding capacity of i-HSA at site II and a relatively lower influence on the binding properties of site I. Ultrafiltration proved to be ineffective in depleting octanoate, while the proposed dialysis protocol, which involves a pH-induced reversible unfolding of the protein, resulted in a total clearance of both stabilizers, confirmed by the full restoration of the binding properties of HSA at all binding sites. The outcomes of this study proved that CD spectroscopy is a suitable technique to evaluate the binding properties of i-HSA, ensuring an assessment of the availability of the binding sites and the possibility of monitoring the clearance of stabilizers. Eventually, the proposed method for their depletion might constitute a connection bridge between albumin in vitro studies and its clinical applications.Graphical abstractHighlightsThe binding capacity of pharmaceutical-grade HSA was assessed by CD spectroscopy.Binding parameters (Symbol, Symbol) were determined for site-specific ICD markers.The binding properties of HSA at site I and site II resulted affected by octanoate.Full depletion of stabilizers was achieved by dialysis involving HSA unfolding.CD analysis proved ideal to monitor the availability of the binding sites of HSA.