Analysis of physiological functions of different human serum albumin pharmaceutical preparations

Introduction

New information is emerging as a basis for reconsidering albumin as a key homeostatic molecule in the critically ill. We are only beginning to understand the full spectrum of albumin properties and action in healthy individuals and hypoalbuminaemic patients. Besides its function in the regulation of colloidal osmotic pressure, albumin has an important antioxidant capacity and a role in the transport of a wide range of drugs, hormones, ions, amino acids, and fatty acids. Few comparative studies have as yet been performed, and they address only a restricted number of parameters mostly defined by the European Pharmacopeia.

Objective

To study and compare the main albumin functions of eight preparations of pharmaceutical-grade albumin, using a battery of different techniques. Two additional albumin preparations, a preparation without stabilisers and a recombinant albumin from Pichia pastoris, were also included in the study.

Methods

The following biochemical and physicochemical parameters were investigated: total protein concentration (Biuret assay) and albumin antigen (nephelometry); quantitative analysis of contaminating proteins by nephelometry, levels of polymers and fragments by gel filtration chromatography on Superose 6, the binding affinity of exogenous ligands for Sudlow's site I (warfarin) or site II (dansylsarcosine) by steady-state spectrofluorimetry, the reactivity of Cys34 with Ellman's reagent, and the esterase-like activity using p-nitrophenyl acetate as substrate by spectrophotometry.

Results

All pharmaceutical-grade products show a purity ranging from 95% to 108%. The main contaminant proteins are prealbumin, transferrin, α-1 acid glycoprotein, haptoglobulin, and retinol-binding protein. All of them are in conformity with the European Pharmacopeia specifications. The warfarin-binding capacity of the 10 albumin preparations was studied. An average binding constant of 2.6 (± 0.3) × 10⁵ M^-1 (n = 1) was found. The presence of stabilisers reduced the binding of dansylsarcosine significantly (by 27–40%). The esterase-like activity toward p-nitrophenyl acetate and the reactivity of Cys34 differed from product to product. Interesting is the absence of free Cys34 in the recombinant albumin.

Conclusion

Significant differences were observed between the 10 different human albumin preparations, recombinant or not. We confirm that the presence of stabilisers such as tryptophan derivatives significantly reduces the binding capacity of Sudlow's site II. Two important physiological properties of albumin, the esterase-like and antioxidant activities, were also found to be modified to different extents in all pharmaceutical-grade products in comparison with the albumin without stabiliser. The benefits of albumin administration should be considered carefully, taking into account the different functions and properties of albumin.