Preparation Of Human Protein C Concentrate

Objective This study is intended to screen efficient stabilizers on stability of protein C treated with solvent/detergent to inactivated lipid-enveloped viruses, to establish a much more inexpensive method for isolating and purifying protein C from Cohn FractionⅣby combining ion exchange chromatography with immobilized metal affinity chromatography, and to achieve an improved utilization of the valuable source material fresh frozen plasma.Methods Cohn FractionⅣwas dissolved with phosphate buffered saline solution in a ratio of 1:10 for 5h at 4℃, and the suspension was centrifuged at 6500 g for 40 minutes at 4℃. The supernatant was loaded through 0.45μm and 0.22μm filters, sequentially, and then it was treated with TNBP (3.3%, w/w) and Tween-80(11%, w/w) at the ratio of 1:10(w/w) for 6h at 24℃. After ultrafiltration and dialysis, protein C was purified further with ion exchange chromatography and immobilized metal affinity chromatography. Molecular weight characterization of protein C was performed by SDS-PAGE and the specificity of protein C was confirmed by Western Blot. Total protein content was determined by the method of Bradford using coomassie blue G-250 as the staining agent under UV absorption at 595 nm. The activity of protein C was determined using chromogenic substrate assay and one-stage clotting assay referenced against the World Health Organization International Standard.Results During solvent/detergent incubation, less than 15% of protein C activity was decreased as a result of the protection of histidine (0.1mol/L) and human serum albumin (2.5%), whereas almost 70% was decreased in case of no addition of stabilizers. The purified protein C concentrate showed only one protein band on gels by SDS-PAGE, of which the molecular weight was ca.62kD. Comfirmed by Western Blot, the 62kD-protein band was protein C. It was achieved a (32.26±4.79)-fold increase in specific activity of protein C over the initiating material with a (47.62±11.30)% yield at the ion exchange chromatography step, and achieved an additional (36.79±3.72)-fold purification and a (59.61±5.59)% yield at the immobilized metal affinity chromatography step. thus, an overall (136.64±6.82)-fold increase in purity and (28.77±9.45)% yield was obtained; the specific activity of the purified protein C was (11.70±0.89)IU/mg protein.Conclusions The combination of histidine (0.1mol/L) and human serum albumin (2.5%) could extremely stabilize protein C incubated with solvent/detergent to inactivate lipid-enveloped viruses. The combination ofion exchange chromatography and immobilized metal affinity chromatography which is much less expensive could be an efficient method for protein C purification from Cohn Fraction IV. Moreover, it could enhance the utilization rate of fresh frozen plasma.