Search

Background C1-esterase inhibitor (C1-INH) is a protein derived from fresh frozen human plasma and is widely used in the treatment of Hereditary Angioedema. C1-INH product must have high purity with preserved functionality. Objective The aim of the study was to perform extensive characterization of human C1-esterase inhibitor (Celestrase 1) with special emphasis on functionality of the protein by different mechanisms along with its purity and structural elucidation. Design Comparative study. Methods This study describes the purification and characterization of an economically viable, highly pure and efficient human plasma-derived C1-INH prepared from cryopoor plasma by combination of chromatography steps, capable of removing protein contaminants. The purification process includes two orthogonal virus clearance steps -solvent detergent and virus retentive filtration and further, characterized by various biochemical and functional assays along with other commercially available entities. Results The developed process yields 0.75 ± 0.1 vials of C1-INH /L of cryopoor plasma with 44.4 ± 3.6% overall process recovery. Celestrase 1 shows comparability with an existing market comparator, with respect to its purity by different methods. Celestrase 1 proved its functionality in binding irreversibly to the complement protein by classical pathway of the complement system and in the Kallikrein system. Additionally, the antigen-to-biologic activity ratio an indicative of the functionality for Celestrase 1 (0.94) was found comparable to market comparator (0.79). Identity of the product was confirmed by Western blot analysis. The structural analysis of Celestrase 1 was found to be similar to market comparator and exists predominantly in α-helix secondary structure by Far UV Circular Dichroism (CD). Conclusion For the treatment of Hereditary Angioedema (HAE), the current study presents a pure, safe, and functionally efficient product that can meet the therapeutic needs of patients deficient in C1-INH.

BACKGROUND Fibrinogen is an important component of hemostasis and clot formation. Replacement therapy with a human Fibrinogen concentrate is the preferred therapeutic option for patients with congenital or acquired Fibrinogen insufficiency. To make the Fibrinogen available for treatment of such patients, Fibrogen-I®, a lyophilized product purified from pooled human plasma was developed and extensively characterized. The product quality attributes were assessed by different biochemical, functional and structural analytical methods. MATERIAL AND METHODS Functional activity of Fibrinogen in the Fibrogen-I® was demonstrated by determination of its clottable protein activity. Purity profiling of Fibrogen-I® was evaluated by SE-HPLC method. Activation and fibrinolysis markers like D-dimer, plasminogen was determined by automated coagulometer and fibrinopeptide A activity by ELISA. Far UV CD analysis was also performed by CD Spectrometer. RESULTS The product exhibited high purity 89.7% by SE-HPLC. Activation and Fibrinolysis marker proteins in the drug product were negligible. The high purity and integrity of Fibrogen-I® is underlined by the ratio of Fibrinogen activity by Clauss/clottable protein with mean calculated value of 0.9 which is in close proximity with theoretical value 1 indicating fully native Fibrinogen. The results of far-UV CD spectroscopy revealed that Fibrogen-I® exists mostly as a β-sheet secondary structure, which is in accordance with the three-dimensional structure of human Fibrinogen. Downscaling experiments for the two dedicated orthogonal pathogen inactivation steps, ie solvent detergent treatment and dry heat treatment, exhibited pathogen safety. DISCUSSION Fibrogen-I® exhibited specific functional activity, desirable quality attributes, pathogen safety and the ability to be made available to patients requiring fast and effective Fibrinogen replacement.