19-08-2014, 10:30 AM
Human blood plasma contains a number of proteins, which have potential therapeutic and hence economic value. Amongst others, Human serum albumin (HSA), a large protein with a molecular weight of 66.5 kDa, is the most abundant protein present in human plasma, with a concentration as high as 35-40 mg/ml. HSA plays a very important role in maintaining the colloidal osmotic pressure and transporting large organic molecules. Albumin is administered as a therapeutic agent in several clinical conditions such as shock and excessive bleeding. The global demand of HSA for the year 2006 was 400 metric tons with sales totaling 0.9 to 1 billion US dollars.
Human blood plasma contains a number of proteins, which have potential therapeutic and hence economic value. Amongst others, Human serum albumin (HSA), a large protein with a molecular weight of 66.5 kDa, is the most abundant protein present in human plasma, with a concentration as high as 35-40 mg/ml. HSA plays a very important role in maintaining the colloidal osmotic pressure and transporting large organic molecules. Albumin is administered as a therapeutic agent in several clinical conditions such as shock and excessive bleeding. The global demand of HSA for the year 2006 was 400 metric tons with sales totaling 0.9 to 1 billion US dollars. Cohn plasma fractionation method that is being the widely used method for purification of HAS from blood serum suffers from many limitations. The Objective of the work was to develop a cost-effective chromatographic method of capturing and purifying HAS from plasma in a single step to be deployed at the beginning of plasma protein purification scheme.
In the view of this, the work done involved HSA purification by affinity chromatography developed from screening of molecules as pseudo-specific ligands attached to a porous and rigid matrix based on polymethacrylate polymer. The work thus deals with purification of Human Serum Albumin from human plasma by pseudoaffinity chromatography. HSA, being a transport protein binds strongly to large number of molecules such as a variety of drugs, fatty acids and amino acids (Kragh-Hansen et.al., 2002). Different molecules were attached to the different matrices as ligands and the resultant adsorbents were screened for their affinity towards HSA by batch binding studies. The adsorbent which showed best binding results was used for selectivity studies for HSA in presence of polyclonal human IgG as model impurity. The selected adsorbent showed good selectivity for HSA as compared to IgG.
This affinity adsorbent, called AlbuSep for which a patent is being filed, was found to exhibit better binding capacity as well as selectivity for HSA when attached to the matrix via a spacer arm. Thus the spacer arm incorporated pseudoaffinity adsorbent was chosen for further work on purification of HSA.
Adsorption isotherm was determined for the adsorption of HSA on pseudoaffinity adsorbent, which gave a Kd (dissociation constant) of the order of 10-6 M and a good Qmax (static binding capacity) of 54 mg/ml. Unsteady state kinetic study was also carried out to get an idea of the uptake rate of the HSA on the adsorbent in order to decide optimum flow rate in column studies.
In summary, the developed adsorbent has the potential to be used for capturing and purification of Human serum albumin (HSA) by chromatographic process. The developed matrix being robust and cost effective can provide a cheaper process for manufacturing HSA.