Process for the preparation of a virus-inactivated FV concentrate starting from human plasma, scalable to industrial level

Inventors

Rossi, Paola • Nardini, Ilaria • Giovacchini, Pierangelo • Mori, Filippo • Farina, Claudio

Assignees

Kedrion SpA

Abstract

The present invention provides a process for purifying FV starting from human plasma or a fractionation intermediate thereof, that is simple, scalable to the industrial level and relatively inexpensive compared to the methods described in the literature to date. The invention consists of the use of two anion exchange chromatography steps, the first of which has the purpose of separating the FV from the PTC component factors, while the second has the purpose of isolating the protein of interest from the majority of plasma proteins by means of selective interaction with the weak anion exchange support used. The process developed has also had a viral inactivation step and a viral removal step included, contributing to the safety of the final product obtained, without however significantly altering the process total recovery of FV, and without necessitating the introduction of additional steps for eliminating the inactivating agents used, thanks to the order in which the various steps are conducted. The process described in the present invention also enables an FV concentrate to be obtained that is stable once frozen at −20° C.

Core Innovation

The invention relates to a process for the purification of intact Factor V (FV) starting from human plasma or an FV-enriched intermediate fraction. The process is designed to obtain intact protein, and it uses two chromatography steps on weak anion exchangers. The first chromatography step is performed in FV non-binding mode, and the second chromatography step is performed in FV capture mode.

A key feature is that the two chromatography steps are separated by at least one viral inactivation step performed by solvent-detergent treatment. After the second chromatography step, the FV-containing solution is subjected to a viral removal step by nanofiltration. The starting material used in the process is cryosupernatant.

The process includes conditioning of cryosupernatant by dilution with Water for injectables (WFI) to obtain a defined conductivity range, and supplementation with the protease inhibitor Aprotinin before contacting the first weak anion exchanger resin. The disclosed approach is associated with improved separation during the first non-binding mode step and downstream viral removal using nanofiltration, together with characterization of FV integrity and identity in the finished product.

Claims Coverage

The document contains one independent claim directed to an intact FV purification process using two weak anion exchanger chromatography steps with FV non-binding and FV capture modes, with viral inactivation between steps and nanofiltration after the second step. Dependent claims refine the weak anion exchanger chemistry and matrix, and add further defined preparation and operating features for the first and second chromatography steps.

Across the independent claim, the inventive concept is anchored in the combination of two weak anion exchanger chromatography modes followed by solvent-detergent viral inactivation and nanofiltration viral removal, using cryosupernatant conditioned with defined conductivity and Aprotinin.

Stated Advantages

Documented Applications