Development of Human Serum Albumin removal kit using solvent mixtures

Abstract

Introduction: Human blood plasma is an informative, complex and easily sampled material. Two important application of plasma include: 1) diagnostic and therapeutic purpose via biomarker discovery and 2) extraction of valuable proteins like human serum albumin (HSA) for medicinal application, which demand is increasing in recent decades. Abundant proteins such as HSA and immunoglobulins (IgGs) mask other proteins and biomarkers. The dynamic range of proteins from picogram to microgram per milliliter caused a big challenge in clinical proteomics, so finding an efficient technique which could achieve two mentioned purpose simultaneously lead to a reduction of treatment process costs noticeably. Aim: The aim of this project was to find a rapid and cost effective method which could reduce abundant proteins such as HSA from human plasma and extract HSA for HSA purification processes. Methods: First, plasma were treated using mono organic solvents, different (NH4)2SO4 saturation levels (V/V %) and binary solvent mixture. After that, plasma treated using mono and chosen di organic solvents in combination with saturated solution of (NH4)2SO4 in various temperature and pH values. The experiments were designed using response surface methodology (RSM) and the consequent models were developed using multivariate regression analysis. The pellet and supernatant of the samples were analyzed using SDS-PAGE method, Bradford assay, and Size exclusion chromatography. Results: The results showed that solvent mixtures (e.g. AC:EtOH) in combination with saturated solution of (NH4)2SO4 possesses higher ability to proper protein precipitating from plasma samples comparing to mono-solvents and (NH4)2SO4 alone. Conclusion: The results showed that by the application of a suitable method for designing of experiments, achievement of the desirable level of purification for HSA is easier, faster and cost-effective. Comparison between the electrophoretic profile of the developed method and commercial products shows, HSA was removed more efficiently using the newly developed method.