title: Purification of fibrinogen and virus removal using preparative electrophoresis.
abstract: The Gradiflow is a novel, scalable preparative electrophoresis technique that uses the dual characteristics of size and charge to isolate target macro- and micromolecules from complex biological solutions. It does this with high resolution and in rapid time. The mild buffers are used to assist in retaining biological activity of the isolated protein. Gradiflow technology employs a sandwich of three polyacrylamide membranes configured to allow passage of macromolecules ranging in size from 10 kDa to 1,500 kDa. Fibrinogen was isolated from cryoprecipitate 1 using a single phase process. This separation was achieved within three hours with yields of 85%. Purified fibrinogen was then characterized using biophysical characterization of fibrin clot structure and compared with clots derived from a commercially available product and human plasma. Significantly, clots developed from Gradiflow fibrinogen had characteristics closer to human plasma. Viral removal characteristics of the Gradiflow were investigated by spiking the source material (cryoprecipitate 1) with canine parvovirus and testing for its presence in the isolated fibrinogen using PCR. Parvo removal was found to be greater than 4 logs and was achieved during the purification process. The Gradiflow offers the advantage of large-scale separation of macromolecules and provides a new approach to fibrinogen separation that is quite distinct from other present-day technologies. The technology is capable of isolating protein with high purity, recovery, and functionality in combination with the removal of viruses during the purification. Furthermore, it is capable of integrating into present production systems, significantly improving yield and functionality of target molecules.
authors.affiliation: Gradipore Ltd, Frenchs Forest, New South Wales, Australia.
authors.forename: A
authors.initials: A
authors.lastname: Gilbert
authors.forename: M
authors.initials: M
authors.lastname: Evtushenko
authors.forename: H
authors.initials: H
authors.lastname: Nair
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005340:Fibrinogen / Q000302:isolation & purification*; D006801:Humans; D017992:Parvovirus / Q000302:isolation & purification*
chemical_list: D017931:DNA Primers; D005340:Fibrinogen