Safety evaluation of a recombinant plasmin derivative lacking kringles 2-5 and rt-PA in a rat model of transient ischemic stroke.

Background: Tissue type plasminogen activator is the only approved thrombolytic agent for the treatment of ischemic stroke. However, it carries the disadvantage of a 10-fold increase in symptomatic and asymptomatic intracranial hemorrhage. A safer thrombolytic agent may improve patient prognosis and increase patient participation in thrombolytic treatment.

Intra-arterial administration of recombinant tissue-type plasminogen activator (rt-PA) causes more intracranial bleeding than does intravenous rt-PA in a transient rat middle cerebral artery occlusion model.

Background: Intra-arterial (IA) administration of rt-PA for ischemic stroke has the potential for greater thrombolytic efficacy, especially for a large thrombus in the M1 or M2 segment of the middle cerebral artery (MCA). Intracranial hemorrhage (ICH) is a concern with IA or intravenous (IV) administration especially as the therapeutic window is extended. However, because IA administration delivers a higher local concentration of agent, the incidence and severity of ICH may be greater than with similar doses IV.

Haemostatic safety of a unique recombinant plasmin molecule lacking kringles 2-5.

We previously demonstrated a significant margin of haemostatic safety for full-length plasmin in comparison with tissue plasminogen activator (t-PA). We now report studies that compare haemostatic safety of full-length plasmin with a novel recombinant plasmin derivative, (Δ K2-5) plasmin, consisting of kringle 1 linked to the serine protease domain of plasmin. Agent was administered intravenously in a randomised, blinded manner in a rabbit model of fibrinolytic haemorrhage.

Simplified recombinant plasmin: production and functional comparison of a novel thrombolytic molecule with plasma-derived plasmin.

A simplified and fully functional deletion mutant of plasminogen was created in which the middle portion of the molecule was removed, resulting in kringle 1 attachment to the serine protease domain. This recombinant plasminogen deletion mutant, Delta(K2-K5)Pg, was produced in the form of inclusion bodies at the yield of up to 200 mg/l in an Escherichia coli T7 expression system. Following protein refolding and purification on lysine-Sepharose, the conversion of the recombinant molecule Delta(K2-K5)Pg to the active enzyme mutant Delta(K2-K5)Pm by plasminogen activators was evaluated, and functional characteristics of the simplified plasmin were studied.

Clearance of prions during plasma protein manufacture.

Protein products isolated from human plasma are an important class of therapeutics that are used to treat patients afflicted with hereditary deficiencies, trauma, and severe infections. Because of the human origin of the starting material for the production of these biological products, there is a risk of transmitting infectious agents, including viruses and the infectious agents that cause transmissible spongiform encephalopathies (TSEs). The agent that is thought to cause TSEs is a disease-associated, misfolded form of the prion protein or PrP(Sc).

An improved Western blot assay to assess the clearance of prion protein from plasma-derived therapeutic proteins.

Specific detection of the pathogenic prion protein, PrP(Sc), is essential for determining the prion clearance capacity of purification processes for therapeutic proteins. Use of a previously described indirect (two-antibody) Western blot assay sometimes resulted in the appearance of non-specific protein bands that interfered with the detection of small amounts of PrP(Sc)-specific signal, limiting the amount of clearance that could be determined for steps so affected. It is shown that these non-specific signals are due to the interaction between immunoglobulin fragments in the sample and the secondary antibody used in the assay.

Partitioning of human and sheep forms of the pathogenic prion protein during the purification of therapeutic proteins from human plasma.

Background: Therapeutic proteins derived from human plasma and other biologic sources have demonstrated an excellent safety record relative to the potential threat of transmissible spongiform encephalopathy (TSE) transmission. Previously, hamster-adapted scrapie was used as a model agent to assess TSE clearance in purification steps leading to the isolation of biopharmaceutical proteins. The current study investigated the validity of hamster scrapie as a model for human TSE clearance studies.

Solvent-dependent precipitation of prion protein.

The misfolded isoform of the prion protein (PrP(Sc)) possesses many unusual physiochemical properties. Previously, we and others reported on the differential partitioning of PrP(Sc) from plasma derived therapeutic proteins during their purification processes. To understand the driving force behind these partitioning differences, we investigated the effects of various solvent conditions on the precipitation of PrP(Sc).