Chromatographic purification and properties of a therapeutic human protein C concentrate

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Vol. 790; no. 1; pp. 199 - 207
• Main Authors: Radosevich, M., Zhou, F.-L., Huart, J.-J., Burnouf, T.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 25.06.2003; Elsevier Science
• Subjects: Aminoacids, peptides. Hormones. Neuropeptides; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Chromatography, Liquid - methods; Electrophoresis, Polyacrylamide Gel; Factor II; Factor IX; Factor VII; Fundamental and applied biological sciences. Psychology; Humans; Mice; Protein C; Protein C - chemistry; Protein C - isolation & purification; Proteins; Rats; Proteins; Protein C; Factor II; Factor IX; Factor VII; Human; Biological fluid; Purification; Gel electrophoresis; Chemical stability; Screening test; In vitro; Chromatography; Blood plasma; In vivo; Adsorption; Animal
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/S1570-0232(03)00091-6

Protein C deficiency (inherited and acquired) has a relatively high incidence rate in the general population worldwide. For many years, protein C deficient patients have been treated with fresh frozen plasma, prothrombin complex concentrates, heparin or oral anticoagulants, which all have clinical drawbacks. We report the production process of a highly purified human protein C concentrate from 1500 l of cryo-poor plasma by a four-step chromatographic procedure. After DEAE-Sephadex adsorption, protein C was separated from clotting factors II, VII and IX by DEAE-Sepharose FF and further purified, using a new strategy, by an on-line chromatographic system combining DMAE-Fractogel and heparin-Sepharose CL-6B. In addition, the product was treated against viral risks by solvent-detergent and nanofiltration on 15-nm membranes. The protein C concentrate was essentially free of other vitamin K-dependent proteins. Proteolytic activity was undetectable. Neither activated protein C, prekallikrein activator, nor activated vitamin K-dependent clotting factors were found resulting in good stability of the protein C activity. In vitro and in vivo animal tests did not reveal any sign of potential thrombogenicity. The final freeze-dried product had a mean protein C concentration of 58 IU/ml and a mean specific activity of 215 IU/mg protein, corresponding to over 12 000-fold purification from plasma. Therefore, this concentrate appears to be of potential benefit for the treatment of protein C deficiency.